Oligomer-conjugate complexes and their use

ABSTRACT

Oligonucleotides, chemically-modified oligonucleotides, and oligonucleotide-conjugate complexes for use in research, diagnostics, and/or therapeutics are described herein. In some embodiments, oligonucleotides comprising a stabilized phosphate moiety covalently attached to the 5′-terminal nucleoside are provided.

CROSS REFERENCED TO RELATED APPLICATIONS

This application is a U.S. National Phase filing under 35 U.S.C. § 371 claiming priority to International Serial No. PCT/US2012/052884 filed Aug. 29, 2012, which claims priority to U.S. Provisional Application 61/528,740, filed Aug. 29, 2011, U.S. Provisional Application 61/532,529, filed Sep. 8, 2011, U.S. Provisional Application 61/535,323, filed Sep. 15, 2011, and U.S. Provisional Application 61/583,963, filed Jan. 6, 2012, each of which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled CORE0098USASEQ_ST25.txt, created on Feb. 24, 2014, which is 16 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Generally, the principle behind antisense technology is that an antisense compound hybridizes to a target nucleic acid and modulates gene expression activities or function, such as transcription or translation. The modulation of gene expression can be achieved by, for example, target degradation or occupancy-based inhibition. An example of modulation of RNA target function by degradation is RNase H-based degradation of the target RNA upon hybridization with a DNA-like antisense compound. Another example of modulation of gene expression by target degradation is RNA interference (RNAi). RNAi generally refers to antisense-mediated gene silencing involving the introduction of dsRNA leading to the sequence-specific reduction of targeted endogenous mRNA levels. An additional example of modulation of RNA target function by an occupancy-based mechanism is modulation of microRNA function. MicroRNAs are small non-coding RNAs that regulate the expression of protein-coding RNAs. The binding of an antisense compound to a microRNA prevents that microRNA from binding to its messenger RNA targets, and thus interferes with the function of the microRNA. Regardless of the specific mechanism, this sequence-specificity makes antisense compounds extremely attractive as tools for target validation and gene functionalization, as well as therapeutics to selectively modulate the expression of genes involved in the pathogenesis of malignancies and other diseases.

Antisense technology is an effective means for reducing the expression of one or more specific gene products and can therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications. Chemically modified nucleosides are routinely used for incorporation into antisense compounds to enhance one or more properties, such as nuclease resistance, pharmacokinetics or affinity for a target RNA. In 1998, the antisense compound, Vitravene® (fomivirsen; developed by Isis Pharmaceuticals Inc., Carlsbad, Calif.) was the first antisense drug to achieve marketing clearance from the U.S. Food and Drug Administration (FDA), and is currently a treatment of cytomegalovirus (CMV)-induced retinitis in AIDS patients.

New chemical modifications have improved the potency and efficacy of antisense compounds, uncovering the potential for oral delivery as well as enhancing subcutaneous administration, decreasing potential for side effects, and leading to improvements in patient convenience. Chemical modifications increasing potency of antisense compounds allow administration of lower doses, which reduces the potential for toxicity, as well as decreasing overall cost of therapy. Modifications increasing the resistance to degradation result in slower clearance from the body, allowing for less frequent dosing. Different types of chemical modifications can be combined in one compound to further optimize the compound's efficacy.

SUMMARY OF THE INVENTION

Provided herein are modified nucleosides, analogs thereof and oligomeric compounds prepared therefrom. In certain embodiments, a single 5′-modified nucleoside or analog thereof is linked to the terminus of an oligomeric compound, preferably at the 5′-terminus. In certain embodiments, oligomeric compounds with conjugate groups are described. In certain embodiments, the oligomeric compounds provided herein are expected to have enhanced nuclease stability. In certain embodiments, the oligomeric compounds and compositions provided herein are expected to hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA. In certain embodiments, the oligomeric compounds provided herein are expected to be useful as primers and probes in diagnostic applications.

The variables are defined individually in further detail herein. It is to be understood that the 5′-modified nucleosides, analogs thereof and oligomeric compounds prepared therefrom as provided herein include all combinations of the embodiments disclosed and variables defined herein.

The present disclosure provides the following non-limiting numbered embodiments:

EMBODIMENT 1

A compound comprising an oligonucleotide consisting of 10-30 linked nucleosides and at least one conjugate group.

EMBODIMENT 2

The compound of embodiment 1, wherein the oligomeric compound comprises a stabilized phosphate moiety covalently attached to the 5′-terminal nucleoside.

EMBODIMENT 3

The compound of embodiment 2, wherein the stabilized phosphate moiety comprises a phosphorus-carbon bond.

EMBODIMENT 4

The compound of embodiment 3, wherein the stabilized phosphate moiety is attached to the 5′-terminal nucleoside via a phosphorus-carbon bond.

EMBODIMENT 5

The compound of any of embodiments 1-4, wherein the stabilized phosphate moiety comprises the following formula:

wherein:

-   -   R_(a) and R_(c) are each, independently, OH, SH, C₁-C₆ alkyl,         substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy,         amino or substituted amino; and     -   R_(b) is O or S.

EMBODIMENT 6

The compound of any of embodiments 1-5, wherein the stabilized phosphate moiety comprises the following formula:

wherein:

-   -   R_(a) and R_(c) are each, independently, OH, SH, C₁-C₆ alkyl,         substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy,         amino or substituted amino;

R_(b) is O or S; and

-   -   X is C(R₁)(R₂) wherein R₁ and R₂ are independently selected from         among: H, halogen, OH, SH, C₁-C₆ alkyl, substituted C₁-C₆ alkyl,         C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, amino or substituted         amino.

EMBODIMENT 7

The compound of any of embodiments 1-6, wherein the stabilized phosphate moiety comprises the following formula:

wherein:

-   -   R_(a) and R_(c) are each, independently, OH, SH, C₁-C₆ alkyl,         substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy,         amino or substituted amino;

R_(b) is O or S;

X is C(R₁)(R₂) and;

Y is selected from C(R₃)(R₄), S, and N; wherein

-   -   R₁ and R₃ are independently selected from among: H, halogen, OH,         SH, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy,         substituted C₁-C₆ alkoxy, amino or substituted amino or R₁ and         R₃ together form a bond; and     -   R₂ and R₄ are independently selected from among: H, halogen, OH,         SH, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy,         substituted C₁-C₆ alkoxy, amino or substituted amino or R₂ and         R₄ together form a bond.

EMBODIMENT 8

The compound of any of embodiments 1 to 7, wherein the 5′-terminal nucleoside has a structure represented by Formula I below:

wherein:

T₁ is a phosphorus moiety;

T₂ is an internucleoside linking group linking the compound of Formula I to the remainder of the oligomeric compound;

M₁ is H, OH or OR₁;

M₂ is OH, OR₁ or N(R₁)(R₂);

each R₁ and R₂ is, independently, C₁-C₆ alkyl, substituted C₁-C₆ alkyl;

r is 0 or 1;

A is has one of the formulas:

Q₁ and Q₂ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy or N(R₃)(R₄);

Q₃ is O, S, N(R₅) or C(R₆)(R₇);

each R₃, R₄, R₅, R₆ and R₇ is, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl or C₁-C₆ alkoxy;

M₃ is O, S, NR₁₄, C(R₁₅)(R₁₆), C(R₁₅)(R₁₆)C(R₁₇)(R₁₈), C(R₁₅)═C(R₁₇), OC(R₁₅)(R₁₆) or OC(R₁₅)(Bx₂);

R₁₄ is H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

R₁₅, R₁₆, R₁₇ and R₁₈ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

one of Bx₁ and Bx₂ is a heterocyclic base moiety and the other of Bx₁ and Bx₂, if present, is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

J₄, J₅, J₆ and J₇ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

or J₄ forms a bridge with either J₅ or J₇ wherein said bridge comprises from 1 to 3 linked biradical groups selected from O, S, NR₁₉, C(R₂₀)(R₂₁), C(R₂₀)═C(R₂₁), C[═C(R₂₀)(R₂₁)] and C(═O) and the other two of J₅, J₆ and J₇ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

each R₁₉, R₂₀ and R₂₁ is, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

G is H, OH, halogen, a conjugate group, or O—[C(R₈)(R₉)]_(n)—[(C═O)_(m)—X₁]_(j)—Z;

each R₈ and R₉ is, independently, H, halogen, C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

X₁ is O, S or N(E₁);

Z is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or N(E₂)(E₃);

E₁, E₂ and E₃ are each, independently, H, C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

n is from 1 to about 6;

m is 0 or 1;

j is 0 or 1;

each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ₁, N(J₁)(J₂), ═NJ₁, SJ₁, N₃, CN, OC(═X₂)J₁, OC(═X₂)N(J₁)(J₂) and C(═X₂)N(J₁)(J₂);

X₂ is O, S or NJ₃;

each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl; and

-   -   when j is 1 then Z is other than halogen or N(E₂)(E₃).

EMBODIMENT 9

The compound of embodiment 8 wherein M₃ is O, CH₂CH₂, CH═CH, OCH₂ or OC(H)(Bx₂) wherein Bx₂ is a heterocyclic base moiety.

EMBODIMENT 10

The compound of any one of embodiments 8 or 9 wherein J₄, J₅, J₆ and J₇ are each H.

EMBODIMENT 11

The compound of any of embodiments 1-10 wherein the 5′-terminal nucleoside has Formula II:

wherein:

Bx is a heterocyclic base moiety;

T₁ is an optionally protected phosphorus moiety;

T₂ is an internucleoside linking group linking the compound of Formula II to the remainder of the oligomeric compound;

A has one of the formulas:

Q₁ and Q₂ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy or N(R₃)(R₄);

Q₃ is O, S, N(R₅) or C(R₆)(R₇);

each R₃, R₄, R₅, R₆ and R₇ is, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl or C₁-C₆ alkoxy;

G is H, OH, halogen, a conjugate group, or O—[C(R₈)(R₉)]_(n)—[(C═O)_(m)—X]_(j)—Z;

each R₈ and R₉ is, independently, H, halogen, C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

X is O, S or N(E₁);

Z is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or N(E₂)(E₃);

E₁, E₂ and E₃ are each, independently, H, C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

n is from 1 to about 6;

m is 0 or 1;

j is 0 or 1;

each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ₁, N(J₁)(J₂), ═NJ₁, SJ₁, N₃, CN, OC(=L)J₁, OC(=L)N(J₁)(J₂) and C(=L)N(J₁)(J₂);

L is O, S or NJ₃;

each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl; and

when j is 1 then Z is other than halogen or N(E₂)(E₃).

EMBODIMENT 12

The compound of any one of embodiments 1 to 11 wherein the 5′-terminal nucleoside has Formula III:

wherein:

Bx is a heterocyclic base moiety;

T₁ is an optionally protected phosphorus moiety;

T₂ is an internucleoside linking group linking the compound of Formula II to the remainder of the oligomeric compound;

A has one of the formulas:

Q₁ and Q₂ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy or N(R₃)(R₄);

Q₃ is O, S, N(R₅) or C(R₆)(R₇);

each R₃, R₄, R₅, R₆ and R₇ is, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl or C₁-C₆ alkoxy;

G is H, OH, halogen, a conjugate group, or O—[C(R₈)(R₉)]_(n)—[(C═O)_(m)—X]_(j)—Z;

each R₈ and R₉ is, independently, H, halogen, C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

X is O, S or N(E₁);

Z is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or N(E₂)(E₃);

E₁, E₂ and E₃ are each, independently, H, C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

n is from 1 to about 6;

m is 0 or 1;

j is 0 or 1;

each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ₁, N(J₁)(J₂), ═NJ₁, SJ₁, N₃, CN, OC(=L)J₁, OC(=L)N(J₁)(J₂) and C(=L)N(J₁)(J₂);

L is O, S or NJ₃;

each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl; and

when j is 1 then Z is other than halogen or N(E₂)(E₃).

EMBODIMENT 13

The compound of any one of embodiments 8-12 wherein A has one of the formulas:

EMBODIMENT 14

The compound of any one of embodiments 8-12 wherein A has the formula:

EMBODIMENT 15

The compound of any one of embodiments 8-12 wherein A has one of the formulas:

EMBODIMENT 16

The compound of any one of embodiments 8-13 wherein Q₁ and Q₂ are each H.

EMBODIMENT 17

The compound of any one of embodiments 8-18 wherein one of Q₁ and Q₂ is H and the other of Q₁ and Q₂ is halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy or substituted C₁-C₆ alkoxy.

EMBODIMENT 18

The compound of any one of embodiments 8-15 wherein one of Q₁ and Q₂ is H and the other of Q₁ and Q₂ is F or CH₃.

EMBODIMENT 19

The compound of any one of embodiments 8-15 wherein Q₁ and Q₂ are each, independently, F or CH₃.

EMBODIMENT 20

The compound of any one of embodiments 8-15 wherein Q₃ is O.

EMBODIMENT 21

The compound of any one of embodiments 8-15 wherein Q₃ is S.

EMBODIMENT 22

The compound of any one of embodiments 8-15 wherein Q₃ is N(R₅).

EMBODIMENT 23

The compound of embodiment 22 wherein R₅ is H.

EMBODIMENT 24

The compound of embodiment 23 wherein R₅ is C₁-C₆ alkyl or substituted C₁-C₆ alkyl.

EMBODIMENT 25

The compound of embodiment 22 wherein R₅ is CH₃.

EMBODIMENT 26

The compound of any one of embodiments 8-15 to wherein Q₃ is C(R₆)(R₇).

EMBODIMENT 27

The compound of embodiment 26 wherein R₆ and R₇ are each H.

EMBODIMENT 28

The compound of embodiment 26 wherein one of R₆ and R₇ is H and the other of R₆ and R₇ is C₁-C₆ alkyl or substituted C₁-C₆ alkyl.

EMBODIMENT 29

The compound of embodiment 26 wherein one of R₆ and R₇ is H and the other of R₆ and R₇ is CH₃.

EMBODIMENT 30

The compound of embodiment 26 wherein R₆ and R₇ are each, independently, C₁-C₆ alkyl, substituted C₁-C₆ alkyl or C₁-C₆ alkoxy.

EMBODIMENT 31

The compound of any one of embodiments 8-12 wherein A has the formula:

EMBODIMENT 32

The compound of any of embodiments 1-31 wherein said 5′-terminal nucleoside has Formula IV:

EMBODIMENT 33

The compound of embodiment 32 wherein Q₁ and Q₂ are each H.

EMBODIMENT 34

The compound of any of embodiments 1 to 42, wherein the 5′-terminal nucleoside has a structure represented by Formula V below:

wherein:

T₁ is an optionally protected phosphorus moiety;

T₂ is an internucleoside linking group linking the compound of Formula I to the remainder of the oligomeric compound;

Z₁C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆ alkyl, substituted C₂-C₆ alkenyl or substituted C₂-C₆ alkynyl;

M₁ is H, OH or OR₁;

M₂ is OH, OR₁ or N(R₁)(R₂);

each R₁ and R₂ is, independently, C₁-C₆ alkyl, substituted C₁-C₆ alkyl;

r is 0 or 1;

each R₃, R₄, R₅, R₆ and R₇ is, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl or C₁-C₆ alkoxy;

M₃ is O, S, NR₁₄, C(R₁₅)(R₁₆), C(R₁₅)(R₁₆)C(R₁₇)(R₁₈), C(R₁₅)═C(R₁₇), OC(R₁₅)(R₁₆) or OC(R₁₅)(Bx₂);

R₁₄ is H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

R₁₅, R₁₆, R₁₇ and R₁₈ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

one of Bx₁ and Bx₂ is a heterocyclic base moiety and the other of Bx₁ and Bx₂, if present, is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

J₄, J₅, J₆ and J₇ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

or J₄ forms a bridge with either J₅ or J₇ wherein said bridge comprises from 1 to 3 linked biradical groups selected from O, S, NR₁₉, C(R₂₀)(R₂₁), C(R₂₀)═C(R₂₁), C[═C(R₂₀)(R₂₁)] and C(═O) and the other two of J₅, J₆ and J₇ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

each R₁₉, R₂₀ and R₂₁ is, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

G is H, OH, halogen, a conjugate group, or O—[C(R₈)(R₉)]_(n)—[(C═O)_(m)—X₁]_(j)—Z;

each R₈ and R₉ is, independently, H, halogen, C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

X₁ is O, S or N(E₁);

Z is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or N(E₂)(E₃);

E₁, E₂ and E₃ are each, independently, H, C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

n is from 1 to about 6;

m is 0 or 1;

j is 0 or 1;

each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ₁, N(J₁)(J₂), ═NJ₁, SJ₁, N₃, CN, OC(═X₂)J₁, OC(═X₂)N(J₁)(J₂) and C(═X₂)N(J₁)(J₂);

X₂ is O, S or NJ₃;

each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl; and

-   -   when j is 1 then Z is other than halogen or N(E₂)(E₃).

EMBODIMENT 35

The compound of embodiment 34 wherein M₃ is O, CH₂CH₂, CH═CH, OCH₂ or OC(H)(Bx₂) wherein Bx₂ is a heterocyclic base moiety.

EMBODIMENT 36

The compound of any one of embodiments 34 or 35 wherein J₄, J₅, J₆ and J₇ are each H.

EMBODIMENT 37

The compound of any one of embodiments 8 to 36 wherein T₁ has the formula:

wherein:

R_(a) and R_(c) are each, independently, protected hydroxyl, protected thiol, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, protected amino or substituted amino; and

R_(b) is O or S.

EMBODIMENT 38

The compound of any one of embodiments 8-36, wherein T₁ has the formula:

wherein:

R_(a) and R_(c) are each a protected hydroxyl; and

R_(b) is O or S.

EMBODIMENT 39

The compound of any one of embodiments 8-36 wherein T₁ has the formula:

wherein:

R_(a) and R_(c) are each, independently, OCH₃, OCH₂CH₃ or CH(CH₃)₂; and

R_(b) is O.

EMBODIMENT 40

The compound of any of embodiments 8-39 wherein Bx is a pyrimidine, substituted pyrimidine, purine or substituted purine.

EMBODIMENT 41

The compound of embodiment 40 wherein Bx is uracil, 5-thiazolo-uracil, thymine, cytosine, 5-methylcytosine, 5-thiazolo-cytosine, adenine, guanine or 2,6-diaminopurine.

EMBODIMENT 42

The compound of any one of embodiments 8-41 wherein G is a conjugate group, halogen, OCH₃, OCH₂F, OCHF₂, OCF₃, OCH₂CH₃, O(CH₂)₂F, OCH₂CHF₂, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—SCH₃, O(CH₂)₂—OCF₃, O(CH₂)₃—N(R₁₀)(R₁₁), O(CH₂)₂—ON(R₁₀)(R₁₁), O(CH₂)₂—O(CH₂)₂—N(R₁₀)(R₁₁), OCH₂C(═O)—N(R₁₀)(R₁₁), OCH₂C(═O)—N(R₁₂)—(CH₂)₂—N(R₁₀)(R₁₁) or O(CH₂)₂—N(R₁₂)—C(═NR₁₃)[N(R₁₀)(R₁₁)] wherein R₁₀, R₁₁, R₁₂ and R₁₃ are each, independently, H or C₁-C₆ alkyl.

EMBODIMENT 43

The compound of any one of embodiments 8-42 wherein G is a conjugate group, halogen, OCH₃, OCF₃, OCH₂CH₃, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—O(CH₂)₂—N(CH₃)₂, OCH₂C(═O)—N(H)CH₃, OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂ or OCH₂—N(H)—C(═NH)NH₂.

EMBODIMENT 44

The compound of any one of embodiments 8-43 wherein G is a conjugate group, F, OCH₃, O(CH₂)₂—OCH₃, a linker group, OCH₂(═O)—N(H)CH₃ or OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂.

EMBODIMENT 45

The compound of any one of embodiments 8-44 wherein G is O(CH₂)₂—OCH₃.

EMBODIMENT 46

The compound of any one of embodiments 8-44 wherein G is F.

EMBODIMENT 47

The compound of any of embodiments 8-44, wherein G is a conjugate group.

EMBODIMENT 48

The compound of any of embodiments 1-47, wherein at least a conjugate group is attached to the oligonucleotide at a nucleoside at position 1, 2, 3, 4, 6, 7, 8, 9, 18, 19, 20, or 21 from the 5′-end of the oligonucleotide or at position 1, 2, 3, 12, 13, 4, 15, 17, 18, 19, 20, or 21 from the 3′-end of the oligonucleotide.

EMBODIMENT 49

The compound of any of embodiments 1-48, comprising a conjugate group attached to any of the 1 to 4 5′-most nucleosides of the oligonucleotide.

EMBODIMENT 50

The compound of any of embodiments 1-49, comprising a conjugate group attached to the 5′-terminal nucleoside of the oligonucleotide.

EMBODIMENT 51

The compound of any of embodiments 1-50, comprising a conjugate group attached to the 2^(nd) nucleoside from the 5′-terminal end of the oligonucleotide.

EMBODIMENT 52

The compound of any of embodiments 1-51, comprising a conjugate group attached to the 3^(rd) nucleoside from the 5′-terminal end of the oligonucleotide.

EMBODIMENT 53

The compound of any of embodiments 1-52, comprising a conjugate group attached to the 4^(th) nucleoside from the 5′-terminal end of the oligonucleotide.

EMBODIMENT 54

The compound of any of embodiments 1-53, comprising a conjugate group attached to any of the 6 to 9 5′-most nucleosides.

EMBODIMENT 55

The compound of any of embodiments 1-54, comprising a conjugate group attached to the 5^(th) nucleoside from the 5′-terminal end of the oligonucleotide.

EMBODIMENT 56

The compound of any of embodiments 1-55, comprising a conjugate group attached to the 6^(th) nucleoside from the 5′-terminal end of the oligonucleotide.

EMBODIMENT 57

The compound of any of embodiments 1-56, comprising a conjugate group attached to the 7^(th) nucleoside from the 5′-terminal end of the oligonucleotide.

EMBODIMENT 58

The compound of any of embodiments 1-57, comprising a conjugate group attached to the 8^(th) nucleoside from the 5′-terminal end of the oligonucleotide.

EMBODIMENT 59

The compound of any of embodiments 1-58, comprising a conjugate group attached to the 9^(th) nucleoside from the 5′-terminal end of the oligonucleotide.

EMBODIMENT 60

The compound of any of embodiments 1-59, comprising a conjugate group attached to the 10^(th) nucleoside from the 5′-terminal end of the oligonucleotide.

EMBODIMENT 61

The compound of any of embodiments 1-60, comprising a conjugate group attached to the 11^(th) nucleoside from the 5′-terminal end of the oligonucleotide.

EMBODIMENT 62

The compound of any of embodiments 1-61, comprising a conjugate group attached to the 12^(th) nucleoside from the 5′-terminal end of the oligonucleotide.

EMBODIMENT 63

The compound of any of embodiments 1-62, comprising a conjugate group attached to the 13^(th) nucleoside from the 5′-terminal end of the oligonucleotide.

EMBODIMENT 64

The compound of any of embodiments 1-63, comprising a conjugate group attached to the 14^(th) nucleoside from the 5′-terminal end of the oligonucleotide.

EMBODIMENT 65

The compound of any of embodiments 1-64, comprising a conjugate group attached to the 15^(th) nucleoside from the 5′-terminal end of the oligonucleotide.

EMBODIMENT 66

The compound of any of embodiments 1-65, comprising a conjugate group attached to the 16^(th) nucleoside from the 5′-terminal end of the oligonucleotide.

EMBODIMENT 67

The compound of any of embodiments 1-66, comprising a conjugate group attached to the 17^(th) nucleoside from the 5′-terminal end of the oligonucleotide.

EMBODIMENT 68

The compound of any of embodiments 1-67, comprising a conjugate group attached to any of the 18 to 21 5′-most phosphate stabilized nucleosides.

EMBODIMENT 69

The compound of any of embodiments 1-68, comprising a conjugate group attached to the 18^(th) nucleoside from the 5′-terminal end of the oligonucleotide.

EMBODIMENT 70

The compound of any of embodiments 1-69, comprising a conjugate group attached to the 19^(th) nucleoside from the 5′-terminal end of the oligonucleotide.

EMBODIMENT 71

The compound of any of embodiments 1-70, comprising a conjugate group attached to the 20^(th) nucleoside from the 5′-terminal end of the oligonucleotide.

EMBODIMENT 72

The compound of any of embodiments 1-71, comprising a conjugate group attached to the 21^(st) nucleoside from the 5′-terminal end of the oligonucleotide.

EMBODIMENT 73

The compound of any of embodiments 1-72, comprising a conjugate group attached to the 2^(nd) nucleoside from the 3′-terminal end of the oligonucleotide.

EMBODIMENT 74

The compound of any of embodiments 1-73, comprising a conjugate group attached to the 3^(rd) nucleoside from the 3′-terminal end of the oligonucleotide.

EMBODIMENT 75

The compound of any of embodiments 1-74, comprising a conjugate group attached to any of the 12^(th) to 21^(st) nucleoside from the 3′-terminal end of the oligonucleotide.

EMBODIMENT 76

The compound of any of embodiments 1-75, comprising a conjugate group attached to the 12^(th) nucleoside from the 3′-terminal end of the oligonucleotide.

EMBODIMENT 77

The compound of any of embodiments 1-76, comprising a conjugate group attached to the 13^(th) nucleoside from the 3′-terminal end of the oligonucleotide.

EMBODIMENT 78

The compound of any of embodiments 1-77, comprising a conjugate group attached to the 14^(th) nucleoside from the 3′-terminal end of the oligonucleotide.

EMBODIMENT 79

The compound of any of embodiments 1-78, comprising a conjugate group attached to the 15^(th) nucleoside from the 3′-terminal end of the oligonucleotide.

EMBODIMENT 80

The compound of any of embodiments 1-79, comprising a conjugate group attached to the 16^(th) nucleoside from the 3′-terminal end of the oligonucleotide.

EMBODIMENT 81

The compound of any of embodiments 1-80, comprising a conjugate group attached to the 17^(th) nucleoside from the 3′-terminal end of the oligonucleotide.

EMBODIMENT 82

The compound of any of embodiments 1-81, comprising a conjugate group attached to the 18^(th) nucleoside from the 3′-terminal end of the oligonucleotide.

EMBODIMENT 83

The compound of any of embodiments 1-82, comprising a conjugate group attached to the 19^(th) nucleoside from the 3′-terminal end of the oligonucleotide.

EMBODIMENT 84

The compound of any of embodiments 1-83, comprising a conjugate group attached to the 20^(th) nucleoside from the 3′-terminal end of the oligonucleotide.

EMBODIMENT 85

The compound of any of embodiments 1-84, comprising a conjugate group attached to the 21^(st) nucleoside from the 3′-terminal end of the oligonucleotide.

EMBODIMENT 86

The compound of any of embodiments 1-85, wherein the 3′-terminal nucleoside does not comprise a conjugate group.

EMBODIMENT 87

The compound of any of embodiments 1-86, wherein the conjugate group is attached at the 2′-position of the nucleoside.

EMBODIMENT 88

The compound of any of embodiments 1-87, wherein the conjugate group is attached at the nucleobase of the nucleoside.

EMBODIMENT 89

The compound of any of embodiments 1-88, wherein the conjugate group is attached at phosphate linkage of the nucleoside

EMBODIMENT 90

The compound of any of embodiments 1-89, wherein the oligonucleotide comprises at least two conjugate groups, wherein at least two conjugate groups are attached to different nucleosides.

EMBODIMENT 91

The compound of any of embodiments 1-90, wherein the oligonucleotide consists of 17 linked nucleosides.

EMBODIMENT 92

The compound of any of embodiments 1-90, wherein the oligonucleotide consists of 18 linked nucleosides.

EMBODIMENT 93

The compound of any of embodiments 1-90, wherein the oligonucleotide consists of 19 linked nucleosides.

EMBODIMENT 94

The compound of any of embodiments 1-90, wherein the oligonucleotide consists of 20 linked nucleosides.

EMBODIMENT 95

The compound of any of embodiments 1-90, wherein the oligonucleotide consists of 21 linked nucleosides.

EMBODIMENT 96

The compound of any of embodiments 1-90, wherein the oligonucleotide consists of 22 linked nucleosides.

EMBODIMENT 97

The compound of any of embodiments 1-90, wherein the oligonucleotide consists of 23 linked nucleosides.

EMBODIMENT 98

The compound of any of embodiments 1-90, wherein the oligonucleotide consists of 24 linked nucleosides groups.

EMBODIMENT 99

The compound of any of embodiments 1-98, wherein each nucleoside is a modified nucleoside.

EMBODIMENT 100

The compound of any of embodiments 1-99, comprising at least one 2′-modified nucleosides.

EMBODIMENT 101

The compound of any of embodiments 1-100, wherein each nucleoside comprises 2′-substituent independently selected from among: 2′-F, 2′-OMe, 2′-MOE, and a conjugate group.

EMBODIMENT 102

The compound of any of embodiments 1-101, wherein each of the 1 to 5 3′-most nucleosides are stabilizing nucleosides.

EMBODIMENT 103

The compound of embodiment 102 wherein the stabilizing nucleosides are 2′-modified nucleosides.

EMBODIMENT 104

The compound of embodiment 103 wherein the stabilizing nucleosides are 2′-MOE modified nucleosides.

EMBODIMENT 105

The compound of any of embodiments 102-104, wherein each of the 2 to 4 3′-most nucleosides are stabilizing nucleosides.

EMBODIMENT 106

The compound of any of embodiments 102-104, wherein each of the 2 3′-most nucleosides are stabilizing nucleosides.

EMBODIMENT 107

The compound of any of embodiments 1 to 106, wherein the 2 3′-most nucleosides are 2′-MOE modified nucleosides and the remaining nucleosides comprise 2-substituents selected from among: 2′-F, 2′-OMe, and a conjugate group.

EMBODIMENT 108

The compound of any of embodiments 1 to 107, wherein the nucleobase of each of the 1 to 5 3′-most terminal nucleosides is a purine.

EMBODIMENT 109

The compound of embodiment 108, wherein the nucleobase of each of the 1 to 5 3′-most terminal nucleoside is an adenine.

EMBODIMENT 110

The compound of any of embodiments 1 to 108, wherein the nucleobase of each of the 2 3′-most terminal nucleosides is a purine.

EMBODIMENT 111

The compound of embodiment 110, wherein the nucleobase of each of the 2 3′-most terminal nucleoside is an adenine.

EMBODIMENT 112

The compound of any of embodiments 1 to 111 wherein each internucleoside linking group is, independently, a phosphodiester internucleoside linking group or a phosphorothioate internucleoside linking group.

EMBODIMENT 113

The compound of any of embodiments 1 to 112 wherein each of the 6 to 9 3′-most internucleoside linkages is a phosphorothioate linkage.

EMBODIMENT 114

The compound of any of embodiments 1 to 113 wherein each of the 6 to 9 3′-most internucleoside linkages is a phosphorothioate linkage and each of the other internucleoside linkages is a phosphodiester linkage.

EMBODIMENT 115

The compound of any of embodiments 1 to 113 wherein each of the 6 to 9 3′-most internucleoside linkages is a phosphorothioate linkage and each of the other internucleoside linkages is a phosphodiester or a phosphorothioate linkage.

EMBODIMENT 116

The compound of any of embodiments 112-115 comprising a linkage alternating region wherein the linkages within the linkage alternating region alternate between phosphodiester linkages and phosphorothioate linkages.

EMBODIMENT 117

The compound of embodiment 116, wherein the linkage alternating region comprises at least 5 linkages.

EMBODIMENT 118

The compound of any of embodiments 1 to 117 wherein each of the 7 3′-most internucleoside linkages is a phosphorothioate linkage.

EMBODIMENT 119

The compound of any of embodiments 1 to 118 wherein each of the 7 3′-most internucleoside linkages is a phosphorothioate linkage, wherein the internucleoside linkage between the 5′-terminal nucleoside and the 2^(nd) nucleoside from the 5′-end is a phosphorothioate linkage, and wherein each of the remaining internucleoside linkages are alternate between phosphorothioate linkages and phosphodiester linkages.

EMBODIMENT 120

The compound of any of embodiments 1-119 having the motif: (P)-s-(A-s-B-o-A)_(x)(-s-B)_(y)-(A-s-B-s-A)_(Z)(-s-B)_(q)-s-(D)-(s-D)_(r)

wherein,

P is a 5′-terminal nucleoside comprising a stabilized phosphate;

each A is independently, either a nucleoside of a first type or a nucleoside that comprises a conjugate group;

each B is independently, either a nucleoside of a second type or a nucleoside that comprises a conjugate group;

each D is independently, either a nucleoside of a third type or a nucleoside that comprises a conjugate group;

wherein the oligonucleotide comprises at least one conjugate group;

s is a phosphorothioate linkage;

o is a phosphodiester linkage;

X is 1-8; and

Y is 1 or 0;

Z is 1-5;

q is 1 or 0; and

r is 0-3.

EMBODIMENT 121

The compound of embodiment 120, wherein each A that does not comprise a conjugate is a 2′-F nucleoside.

EMBODIMENT 122

The compound of any of embodiments 120 to 121, wherein each B that does not comprise a conjugate is a 2′-OMe nucleoside.

EMBODIMENT 123

The compound of any of embodiments 120 to 122, wherein each D that does not comprise a conjugate is a 2′-MOE nucleoside.

EMBODIMENT 124

The compound of any of embodiments 120 to 123, wherein P comprises a conjugate group.

EMBODIMENT 125

The compound of any of embodiments 120 to 123, wherein P comprises a 2′-conjugate group.

EMBODIMENT 126

The compound of any of embodiments 120 to 123, wherein P comprises a 2′-MOE.

EMBODIMENT 127

The compound of any of embodiments 120 to 126, wherein one A comprises a conjugate group.

EMBODIMENT 128

The compound of any of embodiments 120 to 126, wherein one B comprises a conjugate group.

EMBODIMENT 129

The compound of any of embodiments 120 to 128, wherein one D comprises a conjugate group.

EMBODIMENT 130

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a dye.

EMBODIMENT 131

The compound of embodiment 130, wherein the dye is selected from among an acridine dye, a coumarine dye, a rhodamine dye, a xanthene dye, a cyanine dye and a pyrene dye, Texas Red, Alexa Fluor® dye, BODLPY® dye, Fluorescein, Oregon Green® dye, and Rhodamine Green™ dye.

EMBODIMENT 132

The compound of embodiment 130, wherein the dye is selected from an acridine dye, a coumarine dye, a rhodamine dye, a xanthene dye, a cyanine dye, and a pyrene dye.

EMBODIMENT 133

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a targeting moiety.

EMBODIMENT 134

The compound of embodiment 133, wherein the targeting moiety is selected from arginine-glycine-aspartate (RGD) peptide, fibronectin, folate, galactose, an apolipoprotein, insulin, transferrin, a fibroblast growth factor (FOF), an epidermal growth factor (EGF) and an antibody.

EMBODIMENT 135

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a magnetic resonance imaging moiety comprising a paramagnetic compound.

EMBODIMENT 136

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a vitamin.

EMBODIMENT 137

The compound of embodiment 136, wherein the vitamin is a B vitamin.

EMBODIMENT 138

The compound of embodiment 137, wherein the B vitamin is selected from the group consisting of vitamin B₁, vitamin B₂, vitamin B₃, vitamin B₅, vitamin B₆, vitamin B₇, vitamin B₉, vitamin B₁₂.

EMBODIMENT 139

The compound of embodiment 136, wherein the vitamin is a C vitamin.

EMBODIMENT 140

The compound of embodiment 136, wherein the vitamin is a D vitamin.

EMBODIMENT 141

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises an intercalator.

EMBODIMENT 142

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a reporter molecule.

EMBODIMENT 143

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a polyamine.

EMBODIMENT 144

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a polyamide.

EMBODIMENT 145

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises polyethylene glycol.

EMBODIMENT 146

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a thioether.

EMBODIMENT 147

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a polyether.

EMBODIMENT 148

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises cholesterol.

EMBODIMENT 149

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a thiocholesterol.

EMBODIMENT 150

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a cholic acid moiety.

EMBODIMENT 151

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a folate.

EMBODIMENT 152

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a folic acid or folic acid derivative.

EMBODIMENT 153

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises gallic acid or a gallic acid derivative.

EMBODIMENT 154

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a lipid.

EMBODIMENT 155

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a phospholipid.

EMBODIMENT 156

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a fatty acid or fatty acid derivative.

EMBODIMENT 157

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a carbohydrate.

EMBODIMENT 158

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a modified carbohydrate.

EMBODIMENT 159

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a carbohydrate derivative.

EMBODIMENT 160

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a polysaccharide.

EMBODIMENT 161

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises modified polysaccharide.

EMBODIMENT 162

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises mannose.

EMBODIMENT 163

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises galactose.

EMBODIMENT 164

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises mannose derivative.

EMBODIMENT 165

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises galactose derivative.

EMBODIMENT 166

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a modified mannose.

EMBODIMENT 167

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a modified galactose.

EMBODIMENT 168

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a D-mannopyranose.

EMBODIMENT 169

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a L-Mannopyranose.

EMBODIMENT 170

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a D-Arabinose.

EMBODIMENT 171

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a L-Galactose.

EMBODIMENT 172

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a D-xylofuranose.

EMBODIMENT 173

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a L-xylofuranose.

EMBODIMENT 174

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a D-glucose.

EMBODIMENT 175

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a L-glucose.

EMBODIMENT 176

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a D-Galactose.

EMBODIMENT 177

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a L-Galactose.

EMBODIMENT 178

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises α-D-Mannofuranose.

EMBODIMENT 179

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises β-D-Mannofuranose.

EMBODIMENT 180

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises α-D-Mannopyranose

EMBODIMENT 181

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises β-D-Mannopyranose.

EMBODIMENT 182

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises α-D-Glucopyranose.

EMBODIMENT 183

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises β-D-Glucopyranose.

EMBODIMENT 184

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises α-D-Glucofuranose.

EMBODIMENT 185

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises β-D-Glucofuranose.

EMBODIMENT 186

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises α-D-fructofuranose.

EMBODIMENT 187

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises α-D-fructopyranose.

EMBODIMENT 188

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises α-D-Galactopyranose.

EMBODIMENT 189

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises β-D-Galactopyranose.

EMBODIMENT 190

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises α-D-Galactofuranose.

EMBODIMENT 191

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises β-D-Galactofuranose.

EMBODIMENT 192

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises glucosamine.

EMBODIMENT 193

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises sialic acid.

EMBODIMENT 194

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises α-D-galactosamine.

EMBODIMENT 195

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises N-Acetylgalactosamine.

EMBODIMENT 196

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises 2-Amino-3-O—[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose.

EMBODIMENT 197

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises 2-Deoxy-2-methylamino-L-glucopyranose.

EMBODIMENT 198

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises 4,6-Dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose.

EMBODIMENT 199

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises 2-Deoxy-2-sulfoamino-D-glucopyranose

EMBODIMENT 200

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises N-Glycoloyl-α-neuraminic acid.

EMBODIMENT 201

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises 5-thio-β-D-glucopyranose.

EMBODIMENT 202

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises methyl 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside.

EMBODIMENT 203

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises 4-Thio-β-D-galactopyranose.

EMBODIMENT 204

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-gluco-heptopyranoside.

EMBODIMENT 205

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises 2,5-Anhydro-D-allononitrile.

EMBODIMENT 206

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises ribose.

EMBODIMENT 207

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises D-ribose.

EMBODIMENT 208

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises D-4-thioribose.

EMBODIMENT 209

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises L-ribose.

EMBODIMENT 210

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises L-4-thioribose.

EMBODIMENT 211

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises modified ribose.

EMBODIMENT 212

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a ribose derivative.

EMBODIMENT 213

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises biotin.

EMBODIMENT 214

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises phenazine.

EMBODIMENT 215

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises phenanthridine.

EMBODIMENT 216

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises an aromatic compound or an aromatic compound derivative.

EMBODIMENT 217

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises anthraquinone.

EMBODIMENT 218

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises adamantane.

EMBODIMENT 219

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises acridine.

EMBODIMENT 220

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises fluorescein.

EMBODIMENT 221

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a rhodamine.

EMBODIMENT 222

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises coumarin.

EMBODIMENT 223

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises an optical imaging moiety.

EMBODIMENT 224

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a magnetic resonance moiety.

EMBODIMENT 225

The compound of embodiment 162, wherein the magnetic resonance moiety is a paramagnetic compound.

EMBODIMENT 226

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a hydrophobic compound.

EMBODIMENT 227

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a hydrophilic compound.

EMBODIMENT 228

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a peptide.

EMBODIMENT 229

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises an amino acid.

EMBODIMENT 230

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises an amino acid derivative.

EMBODIMENT 231

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a nucleic acid.

EMBODIMENT 232

The compound of embodiments 1 to 129, wherein the conjugate group comprises a nucleic acid derivative.

EMBODIMENT 233

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a heterocycle.

EMBODIMENT 234

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a steroid.

EMBODIMENT 235

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises an ionic complex.

EMBODIMENT 236

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a polyionic complex.

EMBODIMENT 237

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a cationic complex.

EMBODIMENT 238

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises an anionic complex.

EMBODIMENT 239

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a taxane.

EMBODIMENT 240

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises a camptotheca.

EMBODIMENT 241

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises an anthracycline.

EMBODIMENT 242

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises cholesterol.

EMBODIMENT 243

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises palmityl.

EMBODIMENT 244

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises stearoyl.

EMBODIMENT 245

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises lithocholic-oleyl.

EMBODIMENT 246

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₂₂ alkyl.

EMBODIMENT 247

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₂₀ alkyl.

EMBODIMENT 248

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₁₆ alkyl.

EMBODIMENT 249

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₁₀ alkyl.

EMBODIMENT 250

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₂₁ alkyl.

EMBODIMENT 251

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₁₉ alkyl.

EMBODIMENT 252

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₁₈ alkyl.

EMBODIMENT 253

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₁₅ alkyl.

EMBODIMENT 254

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₁₄ alkyl.

EMBODIMENT 255

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₁₃ alkyl.

EMBODIMENT 256

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₁₂ alkyl.

EMBODIMENT 257

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₁₁ alkyl.

EMBODIMENT 258

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₉ alkyl.

EMBODIMENT 259

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₈ alkyl.

EMBODIMENT 260

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₇ alkyl.

EMBODIMENT 261

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₆ alkyl.

EMBODIMENT 262

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₅ alkyl.

EMBODIMENT 263

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₂₂ alkenyl.

EMBODIMENT 264

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₂₀ alkenyl.

EMBODIMENT 265

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₁₆ alkenyl.

EMBODIMENT 266

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₁₀ alkenyl.

EMBODIMENT 267

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₂₁ alkenyl.

EMBODIMENT 268

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₁₉ alkenyl.

EMBODIMENT 269

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₁₈ alkenyl.

EMBODIMENT 270

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₁₅ alkenyl.

EMBODIMENT 271

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₁₄ alkenyl.

EMBODIMENT 272

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₁₃ alkenyl.

EMBODIMENT 273

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₁₂ alkenyl.

EMBODIMENT 274

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₁₁ alkenyl.

EMBODIMENT 275

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₉ alkenyl.

EMBODIMENT 276

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₈ alkenyl.

EMBODIMENT 277

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₇ alkenyl.

EMBODIMENT 278

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₆ alkenyl.

EMBODIMENT 279

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₅ alkenyl.

EMBODIMENT 280

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₂₂ alkynyl.

EMBODIMENT 281

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₂₀ alkynyl.

EMBODIMENT 282

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₁₆ alkynyl.

EMBODIMENT 283

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₁₀ alkynyl.

EMBODIMENT 284

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₂₁ alkynyl.

EMBODIMENT 285

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₁₉ alkynyl.

EMBODIMENT 286

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₁₈ alkynyl.

EMBODIMENT 287

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₁₅ alkynyl.

EMBODIMENT 288

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₁₄ alkynyl.

EMBODIMENT 289

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₁₃ alkynyl.

EMBODIMENT 290

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₁₂ alkynyl.

EMBODIMENT 291

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₁₁ alkynyl.

EMBODIMENT 292

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₉ alkynyl.

EMBODIMENT 293

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₈ alkynyl.

EMBODIMENT 294

The compound of any of, embodiments 1 to 129, wherein the conjugate group comprises C₇ alkynyl.

EMBODIMENT 295

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₆ alkynyl.

EMBODIMENT 296

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises C₅ alkynyl.

EMBODIMENT 297

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises squalene.

EMBODIMENT 298

The compound of any of embodiments 1 to 129, wherein the conjugate group comprises:

EMBODIMENT 299

The compound of any of embodiments 1 to 299, wherein the conjugate group has a pH value of greater than 7.

EMBODIMENT 300

The compound of any of embodiments 1 to 299, wherein the conjugate group has a pH value of less than 7.

EMBODIMENT 301

The compound of any of embodiments 1 to 299, wherein the conjugate group increases the binding affinity to lipoproteins.

EMBODIMENT 302

The compound of any of embodiments 1 to 299, wherein the conjugate group increases the lipophilicity of the oligonucleotide-conjugate complex.

EMBODIMENT 303

The compound of any of embodiments 1 to 299, wherein the conjugate group decreases the lipophilicity of the oligonucleotide-conjugate complex.

EMBODIMENT 304

The compound of any of embodiments 1 to 299, wherein the conjugate group increases the lipophobicity of the oligonucleotide-conjugate complex.

EMBODIMENT 305

The compound of any of embodiments 1 to 299, wherein the conjugate group decreases the lipophobicity of the oligonucleotide-conjugate complex.

EMBODIMENT 306

The compound of any of embodiments 1 to 299, wherein the conjugate group comprises a group that increases lipoprotein binding affinity.

EMBODIMENT 307

The compound of any of embodiments 1 to 299, wherein the conjugate group increases the binding affinity of the oligonucleotide-conjugate complex to albumin.

EMBODIMENT 308

The compound of any of embodiments 1 to 299, wherein the conjugate group decreases the binding affinity of the oligonucleotide-conjugate complex to albumin.

EMBODIMENT 309

The compound of any of embodiments 1 to 299, wherein the conjugate group increases the binding affinity of the oligonucleotide-conjugate complex to glycoproteins.

EMBODIMENT 310

The compound of any of embodiments 1 to 299, wherein the conjugate group decreases the binding affinity of the oligonucleotide-conjugate complex to glycoproteins.

EMBODIMENT 311

The compound of any of embodiments 1 to 299, wherein the conjugate group increases the binding affinity of the oligonucleotide-conjugate complex to α-globulins, β-globulins, and γ-globulins.

EMBODIMENT 312

The compound of any of embodiments 1 to 299, wherein the conjugate group decreases the binding affinity of the oligonucleotide-conjugate complex to α-globulins, β-globulins, and γ-globulins.

EMBODIMENT 313

The compound of any of embodiments 1 to 312, wherein the conjugate group comprises a linker group.

EMBODIMENT 314

The compound of any of embodiments 1 to 313, comprising a linker group selected from the group consisting of a carbamate, N,N-hexyl carbamate, hexanamide, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), 6-aminohexanoic acid (AHEX or AHA), substituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl, and substituted or unsubstituted C₂-C₁₀ allynyl.

EMBODIMENT 315

The compound of any of embodiments 313 to 314, wherein the linker group is hexanamide.

EMBODIMENT 316

The compound of any of embodiments 313 to 314, wherein the linker group is a carbamate.

EMBODIMENT 317

The compound of any of embodiments 313 to 314, wherein the linker group is N,N-hexyl carbamate.

EMBODIMENT 318

The compound of embodiment 313, wherein the linker is an alkyl.

EMBODIMENT 319

The compound of embodiment 313, wherein the linker is an ester.

EMBODIMENT 320

The compound of embodiment 313, wherein the linker is an anhydride.

EMBODIMENT 321

The compound of embodiment 313, wherein the linker is an ether.

EMBODIMENT 322

The compound of embodiment 313, wherein the linker is an amide.

EMBODIMENT 323

The compound of any of embodiments 1 to 322, wherein two or more conjugate groups are attached to the oligomer.

EMBODIMENT 324

The compound of embodiment 323, wherein each conjugate group comprises a linker group.

EMBODIMENT 325

The compound of any of embodiments 323 or 324, wherein at least one conjugate group is selected from the group consisting of cholesterol, palmityl, C₂₂ alkyl, C₂₀ alkyl, C₁₆ alkyl, and C₁₀ alkyl.

EMBODIMENT 326

The compound of any of embodiments 323 or 324, wherein at least one conjugate group comprises cholesterol.

EMBODIMENT 327

The compound of any of embodiments 323 or 324, wherein at least one conjugate group comprises palmityl.

EMBODIMENT 328

The compound of any of embodiments 323 or 324, wherein at least one conjugate group comprises stearoyl.

EMBODIMENT 329

The compound of any of embodiments 323 or 324, wherein at least one conjugate group comprises lithocholic-oleyl.

EMBODIMENT 330

The compound of any of embodiments 323 or 324, wherein at least one conjugate group comprises C₂₂ alkyl.

EMBODIMENT 331

The compound of any of embodiments 323 or 324, wherein at least one conjugate group comprises C₂₀ alkyl.

EMBODIMENT 332

The compound of any of embodiments 323 or 324, wherein at least one conjugate group comprises C₁₆ alkyl.

EMBODIMENT 333

The compound of any of embodiments 323 or 324, wherein at least one conjugate group comprises C₁₀ alkyl.

EMBODIMENT 334

The compound of any of embodiments 1 to 129 or 300 to 333, wherein the conjugate group comprises a structure represented by Formula VIII below:

-   -   wherein:

each J′₁, J′₂, J′₃, J′₄, J′₅, J′₆, J′₇, J′₈, J′₉, and J′₁₀ is, independently, H, halogen, hydroxyl, alcohol, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl, N(A′₁)(A′₂), C(=L′)A′₁, or C(=L′)N(A′₁)(A′₂), C(A′₃)(A′₄), a linker group, or a point of attachment to a linker group, oligomer, or nucleoside;

wherein L′ is O or S;

wherein each A′₁ and A′₂ is, independently, H, halogen, hydroxyl, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, or substituted C₂-C₆ alkynyl; and

wherein each A′₃ and A′₄ is, independently, H, OH, hydroxyl, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl.

EMBODIMENT 335

The compound of embodiment 334, wherein one of J′₁, J′₂, J′₃, J′₄, J′₅, J′₆, J′₇, J′₈, J′₉, and J′₁₀ is H.

EMBODIMENT 336

The compound of embodiment 334, wherein one of J′₁, J′₂, J′₃, J′₄, J′₅, J′₆, J′₇, J′₈, J′₉, and J′₁₀ is C(A′₃)(A′₄).

EMBODIMENT 337

The compound of embodiment 334, wherein J′₆ is a hydroxyl.

EMBODIMENT 338

The compound of embodiment 334, wherein the conjugate group comprises:

EMBODIMENT 339

The compound of any of embodiments 1 to 129 or 300 to 333, wherein at least one conjugate group comprises mannose.

EMBODIMENT 340

The compound of any of embodiments 1 to 129 or 300 to 333, wherein at least conjugate group comprises a carbohydrate cluster.

EMBODIMENT 341

The compound of any of embodiments 1 to 129 or 300 to 333, wherein at least conjugate group comprises a multivalent carbohydrate cluster.

EMBODIMENT 342

The compound of embodiment 340 or 341, wherein at least one of the carbohydrates in the carbohydrate cluster is 2-acetamido-2-deoxy-D-galactopyranose.

EMBODIMENT 343

The compound of embodiment 340 or 341, wherein each of the carbohydrates in the carbohydrate cluster is 2-acetamido-2-deoxy-D-galactopyranose.

EMBODIMENT 344

The compound of embodiment 340 or 341, wherein at least one of the carbohydrates in the carbohydrate cluster comprises galactose.

EMBODIMENT 345

The compound of embodiment 340 or 341, wherein each of the carbohydrates in the carbohydrate cluster comprises galactose.

EMBODIMENT 346

The compound of embodiment 340 or 341, wherein at least one of the carbohydrates in the carbohydrate cluster comprises mannose.

EMBODIMENT 347

The compound of embodiment 340 or 341, wherein each of the carbohydrates in the carbohydrate cluster comprises mannose.

EMBODIMENT 348

The compound of embodiment 340 or 341, wherein at least one of the carbohydrates in the carbohydrate cluster comprises galactosamine.

EMBODIMENT 349

The compound of embodiment 340 or 341, wherein each of the carbohydrates in the carbohydrate cluster comprises galactosamine.

EMBODIMENT 350

The compound of embodiment 340 or 341, wherein at least one of the carbohydrates in the carbohydrate cluster comprises a polysaccharide.

EMBODIMENT 351

The compound of embodiment 340 or 341, wherein each of the carbohydrates in the carbohydrate cluster comprises a polysaccharide.

EMBODIMENT 352

The compound of any of embodiments 1 to 129 or 300 to 333, wherein the conjugate group comprises an amino sugar.

EMBODIMENT 353

The compound of any of embodiments 1 to 129 or 300 to 333, wherein the conjugate group comprises a thio sugar.

EMBODIMENT 354

The compound of any of embodiments 1 to 129 or 300 to 333, wherein the conjugate group comprises:

-   -   wherein L″₄ comprises a linker group;     -   M′ is a scaffold;     -   L″ is a linker group;     -   n is 0 or an integer between 1 and 10;     -   T′ is selected from the group consisting of a carbohydrate, a         modified carbohydrate, and a carbohydrate derivative.

EMBODIMENT 355

The compound of embodiment 354, wherein M′ comprises an ether.

EMBODIMENT 356

The compound of embodiment 354, wherein M′ comprises an anhydride.

EMBODIMENT 357

The compound of embodiment 354, wherein M′ comprises an amide.

EMBODIMENT 358

The compound of embodiment 354, wherein M′ comprises an amine.

EMBODIMENT 359

The compound of embodiment 354, wherein n is 0.

EMBODIMENT 360

The compound of embodiment 354, wherein n is 1.

EMBODIMENT 361

The compound of embodiment 354, wherein n is 2.

EMBODIMENT 362

The compound of embodiment 354, wherein n is 3.

EMBODIMENT 363

The compound of embodiment 354, wherein n is 4.

EMBODIMENT 364

The compound of embodiment 354, wherein n is 5.

EMBODIMENT 365

The compound of embodiment 354, wherein n is 6.

EMBODIMENT 366

The compound of embodiment 354, wherein n is 7.

EMBODIMENT 367

The compound of embodiment 354, wherein n is 8.

EMBODIMENT 368

The compound of embodiment 354, wherein n is 9.

EMBODIMENT 369

The compound of embodiment 354, wherein n is 10.

EMBODIMENT 370

The compound of any of embodiments 1 to 129 or 300 to 333, wherein the conjugate group comprises:

-   -   wherein each L″₁, L″₂, L″₃, and L″₄ comprises a linker group;         and     -   wherein each of T′₁, T′₂, and T′₃ is independently selected from         the group consisting of a targeting agent, a carbohydrate, a         modified carbohydrate, and a carbohydrate derivative.

EMBODIMENT 371

The compound of any of embodiments 1 to 129 or 300 to 333, wherein the conjugate group comprises:

-   -   wherein each L″₁, L″₂, L″₃, and L″₅ comprises a linker group;         and     -   wherein each T′₁, T′₂, and T′₃ is independently selected from         the group consisting of a targeting agent, a carbohydrate, a         modified carbohydrate, and a carbohydrate derivative.

EMBODIMENT 372

The compound of any of embodiments 1 to 129 or 300 to 333, wherein the conjugate group comprises:

-   -   wherein L″₅ comprises a linker group; and     -   wherein each T′₁, T′₂, and T′₃ is independently selected from         the group consisting of a targeting agent, a carbohydrate, a         modified carbohydrate, and a carbohydrate derivative.

EMBODIMENT 373

The compound of any of embodiments 1 to 129 or 300 to 333, wherein the conjugate group comprises:

-   -   wherein L″₄ comprises a linker group; and     -   wherein each T′₁, T′₂, and T′₃ is independently selected from         the group consisting of a targeting agent, a carbohydrate, a         modified carbohydrate, and a carbohydrate derivative.

EMBODIMENT 374

The compound of any of embodiments 1 to 129 or 300 to 333, wherein the conjugate group comprises:

-   -   wherein L″₅ comprises a linker group; and     -   wherein each T′₁, T′₂, and T′₃ is independently selected from         the group consisting of a targeting agent, a carbohydrate, a         modified carbohydrate, and a carbohydrate derivative.

EMBODIMENT 375

The compound of any of embodiments 1 to 129 or 300 to 333, wherein the conjugate group comprises:

-   -   wherein L″₄ comprises a linker group; and     -   wherein each T′₁, T′₂, and T′₃ is independently selected from         the group consisting of a targeting agent, a carbohydrate, a         modified carbohydrate, and a carbohydrate derivative.

EMBODIMENT 376

The compound of any of embodiments 1 to 129 or 300 to 333, wherein the conjugate group comprises:

-   -   wherein L″₅ comprises a linker group; and     -   wherein each T′₁, T′₂, and T′₃ is independently selected from         the group consisting of a targeting agent, a carbohydrate, a         modified carbohydrate, and a carbohydrate derivative.

EMBODIMENT 377

The compound of any of embodiments 1 to 129 or 300 to 333, wherein the conjugate group comprises:

-   -   wherein each T′₁, T′₂, and T′₃ is independently selected from         the group consisting of a targeting agent, a carbohydrate, a         modified carbohydrate, and a carbohydrate derivative.

EMBODIMENT 378

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ comprises a thio sugar.

EMBODIMENT 379

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ comprises an amino sugar.

EMBODIMENT 380

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is mannose.

EMBODIMENT 381

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is galactose.

EMBODIMENT 382

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is D-mannopyranose.

EMBODIMENT 383

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is L-Mannopyranose.

EMBODIMENT 384

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is D-Arabinose.

EMBODIMENT 385

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is L-Galactose.

EMBODIMENT 386

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is D-xylofuranose.

EMBODIMENT 387

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is L-xylofuranose.

EMBODIMENT 388

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is D-glucose.

EMBODIMENT 389

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is L-glucose.

EMBODIMENT 390

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is D-Galactose.

EMBODIMENT 391

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is L-Galactose.

EMBODIMENT 392

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is α-D-Mannofuranose.

EMBODIMENT 393

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is β-D-Mannofuranose.

EMBODIMENT 394

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is α-D-Mannopyranose

EMBODIMENT 395

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is β-D-Mannopyranose.

EMBODIMENT 396

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is α-D-Glucopyranose.

EMBODIMENT 397

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is β-D-Glucopyranose.

EMBODIMENT 398

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is α-D-Glucofuranose.

EMBODIMENT 399

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is β-D-Glucofuranose.

EMBODIMENT 400

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is α-D-fructofuranose.

EMBODIMENT 401

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is α-D-fructopyranose.

EMBODIMENT 402

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is α-D-Galactopyranose.

EMBODIMENT 403

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is β-D-Galactopyranose.

EMBODIMENT 404

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is α-D-Galactofuranose.

EMBODIMENT 405

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is β-D-Galactofuranose.

EMBODIMENT 406

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is glucosamine.

EMBODIMENT 407

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is sialic acid.

EMBODIMENT 408

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is α-D-galactosamine.

EMBODIMENT 409

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is N-Acetylgalactosamine.

EMBODIMENT 410

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is 2-Amino-3-O—[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose.

EMBODIMENT 411

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is 2-Deoxy-2-methylamino-L-glucopyranose.

EMBODIMENT 412

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is 4,6-Dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose.

EMBODIMENT 413

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is 2-Deoxy-2-sulfoamino-D-glucopyranose.

EMBODIMENT 414

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is N-Glycoloyl-α-neuraminic acid.

EMBODIMENT 415

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is 5-thio-β-D-glucopyranose.

EMBODIMENT 416

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is methyl-2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside.

EMBODIMENT 417

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is 4-Thio-β-D-galactopyranose.

EMBODIMENT 418

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-gluco-heptopyranoside.

EMBODIMENT 419

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is 2,5-Anhydro-D-allononitrile.

EMBODIMENT 420

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is pyranose or a pyranose derivative.

EMBODIMENT 421

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, or T′₃ is furanose or a furanose derivative.

EMBODIMENT 422

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, and T′₃ is ribose.

EMBODIMENT 423

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, and T′₃ is D-ribose.

EMBODIMENT 424

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, and T′₃ is D-4-thioribose.

EMBODIMENT 425

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, and T′₃ is L-ribose.

EMBODIMENT 426

The compound of any of embodiments 354 to 378, wherein one of T′₁, T′₂, and T′₃ is L-4-thioribose.

EMBODIMENT 427

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ comprises a thio sugar.

EMBODIMENT 428

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ comprises an amino sugar.

EMBODIMENT 429

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is mannose.

EMBODIMENT 430

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is galactose.

EMBODIMENT 431

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is D-mannopyranose.

EMBODIMENT 432

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is L-Mannopyranose.

EMBODIMENT 433

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is D-Arabinose.

EMBODIMENT 434

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is L-Galactose.

EMBODIMENT 435

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is D-xylofuranose.

EMBODIMENT 436

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is L-xylofuranose.

EMBODIMENT 437

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is D-glucose.

EMBODIMENT 438

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is L-glucose.

EMBODIMENT 439

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is D-Galactose.

EMBODIMENT 440

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is L-Galactose.

EMBODIMENT 441

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is α-D-Mannofuranose.

EMBODIMENT 442

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is β-D-Mannofuranose.

EMBODIMENT 443

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is α-D-Mannopyranose

EMBODIMENT 444

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is β-D-Mannopyranose.

EMBODIMENT 445

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is α-D-Glucopyranose.

EMBODIMENT 446

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is β-D-Glucopyranose.

EMBODIMENT 447

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is α-D-Glucofuranose.

EMBODIMENT 448

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is β-D-Glucofuranose.

EMBODIMENT 449

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is α-D-fructofuranose.

EMBODIMENT 450

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is α-D-fructopyranose.

EMBODIMENT 451

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is α-D-Galactopyranose.

EMBODIMENT 452

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is β-D-Galactopyranose.

EMBODIMENT 453

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is α-D-Galactofuranose.

EMBODIMENT 454

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is β-D-Galactofuranose.

EMBODIMENT 455

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is glucosamine.

EMBODIMENT 456

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is sialic acid.

EMBODIMENT 457

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is α-D-galactosamine.

EMBODIMENT 458

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is N-Acetylgalactosamine.

EMBODIMENT 459

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is 2-Amino-3-O—[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose.

EMBODIMENT 460

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is 2-Deoxy-2-methylamino-L-glucopyranose.

EMBODIMENT 461

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is 4,6-Dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose.

EMBODIMENT 462

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is 2-Deoxy-2-sulfoamino-D-glucopyranose.

EMBODIMENT 463

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is N-Glycoloyl-α-neuraminic acid.

EMBODIMENT 464

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is 5-thio-β-D-glucopyranose.

EMBODIMENT 465

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is methyl 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside.

EMBODIMENT 466

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is 4-Thio-β-D-galactopyranose.

EMBODIMENT 467

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-gluco-heptopyranoside.

EMBODIMENT 468

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is 2,5-Anhydro-D-allononitrile.

EMBODIMENT 469

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is pyranose or a pyranose derivative.

EMBODIMENT 470

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is furanose or a furanose derivative.

EMBODIMENT 471

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is ribose.

EMBODIMENT 472

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is D-ribose.

EMBODIMENT 473

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is D-4-thioribose.

EMBODIMENT 474

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is L-ribose.

EMBODIMENT 475

The compound of any of embodiments 354 to 378, wherein each of T′₁, T′₂, and T′₃ is L-4-thioribose.

EMBODIMENT 476

The compound of any of embodiments 1 to 129 or 300 to 333, wherein the conjugate group comprises:

-   -   wherein each L″₁, L″₂, L″₃, and L″₄ is a linker group; and     -   wherein each R_(j), R_(k), R_(l), R_(m), R_(n), R_(o), R_(p),         R_(q), and R_(s) is independently selected from the group         consisting of H, a protecting group, C₁-C₆ alkyl, substituted         C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆         alkynyl, substituted C₂-C₆ alkynyl, C₁-C₆ alkoxy, substituted         C₁-C₆ alkoxy, amino, and amido.

EMBODIMENT 477

The compound of any of embodiments 1 to 129 or 300 to 333, wherein the conjugate group comprises:

-   -   wherein each L″₁, L″₂, L″₃, and L″₄ is a linker group₅; and     -   wherein each R_(j), R_(k), R_(l), R_(m), R_(n), R_(o), R_(p),         R_(q), and R_(s) is independently selected from the group         consisting of H, a protecting group, C₁-C₆ alkyl, substituted         C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆         alkynyl, substituted C₂-C₆ alkynyl, C₁-C₆ alkoxy, substituted         C₁-C₆ alkoxy, amino, and amido.

EMBODIMENT 478

The compound of any of embodiments 1 to 129 or 300 to 333, wherein the conjugate group comprises:

-   -   wherein L″₅ is a linker group; and     -   wherein each R_(j), R_(k), R_(l), R_(m), R_(n), R_(o), R_(p),         R_(q), and R_(s) is independently selected from the group         consisting of H, a protecting group, C₁-C₆ alkyl, substituted         C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆         alkynyl, substituted C₂-C₆ alkynyl, C₁-C₆ alkoxy, substituted         C₁-C₆ alkoxy, amino, and amido.

EMBODIMENT 479

The compound of any of embodiments 1 to 129 or 300 to 333, wherein the conjugate group comprises:

-   -   wherein L″₅ is a linker group; and     -   wherein each R_(j), R_(k), R_(l), R_(m), R_(n), R_(o), R_(p),         R_(q), and R_(s) is independently selected from the group         consisting of H, a protecting group, C₁-C₆ alkyl, substituted         C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆         alkynyl, substituted C₂-C₆ alkynyl, C₁-C₆ alkoxy, substituted         C₁-C₆ alkoxy, amino, and amido.

EMBODIMENT 480

The compound of any of embodiments 1 to 129 or 300 to 333, wherein the conjugate group comprises:

-   -   wherein L″₅ is a linker group; and     -   wherein each R_(j), R_(k), R_(l), R_(m), R_(n), R_(o), R_(p),         R_(q), and R_(s) is independently selected from the group         consisting of H, a protecting group, C₁-C₆ alkyl, substituted         C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆         alkynyl, substituted C₂-C₆ alkynyl, C₁-C₆ alkoxy, substituted         C₁-C₆ alkoxy, amino, and amido.

EMBODIMENT 481

The compound of any of embodiments 476 to 480, wherein each one of R_(j), R_(k), R_(l), R_(m), R_(n), R_(o), R_(p), R_(q), and R_(s) is H.

EMBODIMENT 482

The compound of any of embodiments 476 to 480, wherein each one of R_(j), R_(k), R_(l), R_(m), R_(n), R_(o), R_(p), R_(q), and R_(s) comprises a protecting group.

EMBODIMENT 483

The compound of embodiment 482, wherein the protecting group comprises an acetate.

EMBODIMENT 484

The compound of any of embodiments 476 to 480, wherein at least one of R_(j), R_(k), R_(l), R_(m), R_(n), R_(o), R_(p), R_(q), and R_(s) is H.

EMBODIMENT 485

The compound of any of embodiments 476 to 480, wherein at least one of R_(j), R_(k), R_(l), R_(m), R_(n), R_(o), R_(p), R_(q), and R_(s) comprises a protecting group.

EMBODIMENT 486

The compound of embodiment 485, wherein the protecting group comprises an acetate.

EMBODIMENT 487

The compound of any of embodiments 1 to 129 or 300 to 333, wherein the conjugate group comprises:

-   -   wherein L″₅ is a linker group.

EMBODIMENT 488

The compound of any of embodiments 1 to 129 or 300 to 333, wherein the conjugate group comprises:

-   -   wherein L″₅ is a linker group.

EMBODIMENT 489

The compound of any of embodiments 1 to 129 or 300 to 333, wherein the conjugate group comprises:

-   -   wherein L″₅ is a linker group.

EMBODIMENT 490

The compound of any of embodiments 1 to 129 or 300 to 333, wherein the conjugate group comprises:

-   -   wherein L″₅ is a linker group.

EMBODIMENT 491

The compound of any of embodiments 354 to 490, wherein each linker group is independently selected from the group consisting of a peptide, an ether, polyethylene glycol, alkyl, C₁-C₂₀ alkyl, substituted C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, substituted C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, substituted C₂-C₂₀ alkynyl, C₁-C₂₀ alkoxy, substituted C₁-C₂₀ alkoxy, amino, amido, pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and 6-aminohexanoic acid.

EMBODIMENT 492

The compound of any of embodiments 354 to 490, wherein each one of L″₁, L″₂, L″₃, L″₄, and L″₅, is independently selected from the group consisting of a peptide, an ether, polyethylene glycol, alkyl, C₁-C₂₀ alkyl, substituted C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, substituted C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, substituted C₂-C₂₀ alkynyl, C₁-C₂₀ alkoxy, substituted C₁-C₂₀ alkoxy, amino, amido, pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and 6-aminohexanoic acid.

EMBODIMENT 493

The compound of any of embodiments 1 to 129 or 300 to 333, wherein the conjugate group comprises:

EMBODIMENT 494

The compound of any of embodiments 1 to 129 or 300 to 333, wherein the conjugate group comprises:

EMBODIMENT 495

The compound of any of embodiments 1 to 129 or 300 to 333, wherein the conjugate group comprises:

EMBODIMENT 496

The compound of any of embodiments 1 to 129 or 300 to 333, wherein the conjugate group comprises:

EMBODIMENT 497

The compound of any of embodiments 1 to 129 or 300 to 333, wherein the conjugate group comprises:

EMBODIMENT 498

The compound of any of embodiments 1 to 129 or 300 to 333, wherein the conjugate group comprises arachidonoyl.

EMBODIMENT 499

The compound of any of embodiments 1 to 323, wherein the conjugate group increases the binding affinity towards higher molecular weight proteins.

EMBODIMENT 500

The compound of any of embodiments 1 to 323, wherein the conjugate group increases the binding affinity towards higher molecular weight plasma proteins.

EMBODIMENT 501

The compound of any of embodiments, 1 to 500 wherein the conjugate group decreases the binding affinity towards higher molecular weight plasma proteins.

EMBODIMENT 502

The compound of any of embodiments, 1 to 500 wherein the conjugate group increases the stability of the compound.

EMBODIMENT 503

The compound of any of embodiments, 1 to 500 wherein the conjugate group increases the concentration of the compound in the liver.

EMBODIMENT 504

The compound of any of embodiments, 1 to 500 wherein the conjugate group increases the affinity of the compound in the liver.

EMBODIMENT 505

The compound of any of embodiments, 1 to 500 wherein the conjugate group modulates the amount or activity of a target nucleic acid in the liver.

EMBODIMENT 506

The compound of any of embodiments, 1 to 500 wherein the conjugate group decreases the amount or activity of a target nucleic acid in the liver.

EMBODIMENT 507

The compound of any of embodiments, 1 to 500 wherein the conjugate group increases the concentration of the compound in adipose tissue.

EMBODIMENT 508

The compound of any of embodiments, 1 to 500 wherein the conjugate group increases the affinity of the compound in adipose tissue.

EMBODIMENT 509

The compound of any of embodiments, 1 to 500 wherein the conjugate group modulates the amount or activity of a target nucleic acid in adipose tissue.

EMBODIMENT 510

The compound of any of embodiments, 1 to 500 wherein the conjugate group decreases the amount or activity of a target nucleic acid in adipose tissue.

EMBODIMENT 511

The compound of any of embodiments 499 to 510, wherein the conjugate comprises a group selected from C₁₆, cholesterol, and C₈.

EMBODIMENT 512

The compound of any of embodiments 499 to 510, wherein the conjugate comprises C₁₆.

EMBODIMENT 513

The compound of any of embodiments 499 to 510, wherein the conjugate comprises cholesterol.

EMBODIMENT 514

The compound of any of embodiments 499 to 510, wherein the conjugate comprises C₈.

EMBODIMENT 515

The compound of any of embodiments 1 to 514, wherein the conjugate group comprises a linker group.

EMBODIMENT 516

The compound of any of embodiments 1 to 514, wherein the conjugate group consists of a conjugate.

EMBODIMENT 517

The compound of any of embodiments 1 to 129 or 300 to 333, wherein the conjugate group comprises a cell penetrating peptide.

EMBODIMENT 518

The compound of any of embodiments 1 to 517, wherein the compound is single-stranded.

EMBODIMENT 519

A double-stranded composition comprising:

-   -   a first oligomeric compound and a second oligomeric compound;         wherein the first oligomeric compound is partially complementary         to the second oligomeric compound and the second oligomeric         compound is complementary to a nucleic acid target; and wherein         -   at least one of the first compound comprises an oligomeric             compound according to any one of embodiments 1 to 496.

EMBODIMENT 520

The double-stranded compound of embodiment 519, wherein the second oligomeric compound comprises a compound according to any one of embodiments 1 to 517.

EMBODIMENT 521

The double-stranded compound of embodiment 499, wherein the first oligomeric compound comprises a compound according to any one of embodiments 1 to 517.

EMBODIMENT 522

A pharmaceutical composition comprising the compound of any one of embodiments 1 to 517 and a pharmaceutically acceptable carrier or diluent.

EMBODIMENT 523

A pharmaceutical composition comprising a therapeutically effective amount of the compound of any one of embodiments 1 to 517 and a pharmaceutically acceptable carrier or diluent.

EMBODIMENT 524

A method of inhibiting protein expression in a cell by contacting the cell with the oligomeric compound according to any one of embodiments 1 to 517 or the pharmaceutical composition of embodiment 522 or 523.

EMBODIMENT 525

A method of treating or ameliorating a disease or condition comprising administering an effective amount of the oligomer compound of any one of embodiments 1 to 516 or the pharmaceutical composition of embodiment 522 or 523.

EMBODIMENT 526

The method of embodiment 525, wherein the disease or condition is selected from the group consisting of cardiovascular disease, metabolic disease, cancer, inflammatory disease, and neurodegenerative disease.

EMBODIMENT 527

Use of the compound of any of embodiments 1 to 517 or the pharmaceutical composition of embodiment 522 or 523, for the preparation of a medicament for treating a disease or condition.

EMBODIMENT 528

Use of the compound of any of embodiments 1 to 517 or the pharmaceutical composition of embodiment 522 or 523, to activate the RISC pathway.

EMBODIMENT 529

Use of the compound of any of embodiments 1 to 517 or the pharmaceutical composition of embodiment 522 or 523, to activate RNAi in a cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the accumulation of C10 conjugated ASOs and ASOs without conjugates in the liver.

FIG. 2 illustrates the accumulation of C16 conjugated ASOs and ASOs without conjugates in the liver.

FIG. 3 illustrates the accumulation of C16 conjugated ASOs and ASOs without conjugates in the liver as determined by ELISA.

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.

A. DEFINITIONS

Unless specific definitions are provided, the nomenclature used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis, and chemical analysis. Certain such techniques and procedures may be found for example in “Carbohydrate Modifications in Antisense Research” Edited by Sangvi and Cook, American Chemical Society, Washington D.C., 1994; “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., 21^(st) edition, 2005; and “Antisense Drug Technology, Principles, Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press, Boca Raton, Fla.; and Sambrook et al., “Molecular Cloning, A laboratory Manual,” 2^(nd) Edition, Cold Spring Harbor Laboratory Press, 1989, which are hereby incorporated by reference for any purpose. Where permitted, all patents, applications, published applications and other publications and other data referred to throughout in the disclosure are incorporated by reference herein in their entirety.

Unless otherwise indicated, the following terms have the following meanings:

As used herein, “nucleoside” means a compound comprising a nucleobase moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (as found in DNA and RNA) and modified nucleosides. Nucleosides may be linked to a phosphate moiety.

As used herein, “chemical modification” means a chemical difference in a compound when compared to a naturally occurring counterpart. Chemical modifications of oligonucleotides include nucleoside modifications (including sugar moiety modifications and nucleobase modifications) and internucleoside linkage modifications. In reference to an oligonucleotide, chemical modification does not include differences only in nucleobase sequence.

As used herein, “furanosyl” means a structure comprising a 5-membered ring comprising four carbon atoms and one oxygen atom.

As used herein, “naturally occurring sugar moiety” means a ribofuranosyl as found in naturally occurring RNA or a deoxyribofuranosyl as found in naturally occurring DNA.

As used herein, “sugar moiety” means a naturally occurring sugar moiety or a modified sugar moiety of a nucleoside.

As used herein, “modified sugar moiety” means a substituted sugar moiety or a sugar surrogate.

As used herein, “substituted sugar moiety” means a furanosyl that is not a naturally occurring sugar moiety. Substituted sugar moieties include, but are not limited to furanosyls comprising substituents at the 2′-position, the 3′-position, the 5′-position and/or the 4′-position. Certain substituted sugar moieties are bicyclic sugar moieties.

As used herein, “2′-substituted sugar moiety” means a furanosyl comprising a substituent at the 2′-position other than H or OH. Unless otherwise indicated, a 2′-substituted sugar moiety is not a bicyclic sugar moiety (i.e., the 2′-substituent of a 2′-substituted sugar moiety does not form a bridge to another atom of the furanosyl ring.

As used herein, “MOE” means —OCH₂CH₂OCH₃.

As used herein, “2′-F nucleoside” refers to a nucleoside comprising a sugar comprising fluorine at the 2′ position. Unless otherwise indicated, the fluorine in a 2′-F nucleoside is in the ribo position (replacing the OH of a natural ribose).

As used herein, “2′-F ANA” refers to a 2′-F substituted nucleoside, wherein the fluoro group is in the arabino position.

As used herein the term “sugar surrogate” means a structure that does not comprise a furanosyl and that is capable of replacing the naturally occurring sugar moiety of a nucleoside, such that the resulting nucleoside sub-units are capable of linking together and/or linking to other nucleosides to form an oligomeric compound which is capable of hybridizing to a complementary oligomeric compound. Such structures include rings comprising a different number of atoms than furanosyl (e.g., 4, 6, or 7-membered rings); replacement of the oxygen of a furanosyl with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); or both a change in the number of atoms and a replacement of the oxygen. Such structures may also comprise substitutions corresponding to those described for substituted sugar moieties (e.g., 6-membered carbocyclic bicyclic sugar surrogates optionally comprising additional substituents). Sugar surrogates also include more complex sugar replacements (e.g., the non-ring systems of peptide nucleic acid). Sugar surrogates include without limitation morpholinos, cyclohexenyls and cyclohexitols.

As used herein, “bicyclic sugar moiety” means a modified sugar moiety comprising a 4 to 7 membered ring (including but not limited to a furanosyl) comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure. In certain embodiments, the 4 to 7 membered ring is a sugar ring. In certain embodiments the 4 to 7 membered ring is a furanosyl. In certain such embodiments, the bridge connects the 2′-carbon and the 4′-carbon of the furanosyl.

As used herein, “nucleotide” means a nucleoside further comprising a phosphate linking group. As used herein, “linked nucleosides” may or may not be linked by phosphate linkages and thus includes, but is not limited to “linked nucleotides.” As used herein, “linked nucleosides” are nucleosides that are connected in a continuous sequence (i.e. no additional nucleosides are present between those that are linked).

As used herein, “nucleobase” means a group of atoms that can be linked to a sugar moiety to create a nucleoside that is capable of incorporation into an oligonucleotide, and wherein the group of atoms is capable of bonding with a complementary naturally occurring nucleobase of another oligonucleotide or nucleic acid. Nucleobases may be naturally occurring or may be modified.

As used herein the terms, “unmodified nucleobase” or “naturally occurring nucleobase” means the naturally occurring heterocyclic nucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) (including 5-methyl C), and uracil (U).

As used herein, “modified nucleobase” means any nucleobase that is not a naturally occurring nucleobase.

As used herein, “modified nucleoside” means a nucleoside comprising at least one chemical modification compared to naturally occurring RNA or DNA nucleosides. Modified nucleosides comprise a modified sugar moiety and/or a modified nucleobase.

As used herein, “bicyclic nucleoside” or “BNA” means a nucleoside comprising a bicyclic sugar moiety.

As used herein, “constrained ethyl nucleoside” or “cEt” means a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH(CH₃)—O-2′ bridge.

As used herein, “locked nucleic acid nucleoside” or “LNA” means a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH₂—O-2′ bridge.

As used herein, “2′-substituted nucleoside” means a nucleoside comprising a substituent at the 2′-position other than H or OH. Unless otherwise indicated, a 2′-substituted nucleoside is not a bicyclic nucleoside.

As used herein, “2′-deoxynucleoside” means a nucleoside comprising 2′-H furanosyl sugar moiety, as found in naturally occurring deoxyribonucleosides (DNA). In certain embodiments, a 2′-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (e.g., uracil).

As used herein, “RNA-like nucleoside” means a modified nucleoside that adopts a northern configuration and functions like RNA when incorporated into an oligonucleotide. RNA-like nucleosides include, but are not limited to 2′-endo furanosyl nucleosides and RNA surrogates.

As used herein, “2′-endo-furanosyl nucleoside” means an RNA-like nucleoside that comprises a substituted sugar moiety that has a 2′-endo conformation. 2′-endo-furanosyl nucleosides include, but are not limited to: 2′-MOE, 2′-F, 2′-OMe, LNA, ENA, and cEt nucleosides.

As used herein, “RNA-surrogate nucleoside” means an RNA-like nucleoside that does not comprise a furanosyl. RNA-surrogate nucleosides include, but are not limited to hexitols and cyclopentanes.

As used herein, “phosphorous moiety” refers to a to monovalent P^(V) phosphorus radical group. In certain embodiments, a phosphorus moiety is selected from: a phosphate, phosphonate, alkylphosphonate, aminoalkyl phosphonate, phosphorothioate, phosphoramidite, alkylphosphonothioate, phosphorodithioate, thiophosphoramidate, phosphotriester and the like.

In certain embodiments, modified phosphorous moieties have the following structural formula:

wherein:

R_(a) and R_(c) are each, independently, OH, SH, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, amino or substituted amino; and

R_(b) is O or S.

The term “phosphate moiety” as used herein, refers to a terminal phosphate group that includes unmodified phosphates (—O—P(═O)(OH)OH) as well as modified phosphates. Modified phosphates include but are not limited to phosphates in which one or more of the O and OH groups is replaced with H, O, S, N(R) or alkyl where R is H, an amino protecting group or unsubstituted or substituted alkyl.

As used herein, “phosphate stabilizing modification” refers to a modification that results in stabilization of a 5′-phosphate moiety of the 5′-terminal nucleoside of an oligonucleotide, relative to the stability of an unmodified 5′-phosphate of an unmodified nucleoside under biologic conditions. Such stabilization of a 5′-phosphate group includes but is not limited to resistance to removal by phosphatases. Phosphate stabilizing modifications include, but are not limited to, modification of one or more of the atoms that binds directly to the phosphorus atom, modification of one or more atoms that link the phosphorus to the 5′-carbon of the nucleoside, and modifications at one or more other positions of the nucleoside that result in stabilization of the phosphate. Phosphate moieties that are stabilized by one or more phosphate stabilizing modification are referred to herein as “stabilized phosphate moieties.”

In certain embodiments, compounds of the present invention comprise oligonucleotides comprising a stabilized phosphate moiety at the 5′-terminus. In certain such embodiments, the phosphorus atom of the stabilized phosphate moiety is attached to the 5′-terminal nucleoside through a phosphorus-carbon bond. In certain embodiments, the carbon of that phosphorus-carbon bond is in turn bound to the 5′-position of the nucleoside.

In certain embodiments, the oligonucleotide optionally comprises a conjugate group and optionally comprises a 5′-stabilized phosphate moiety having the following formula:

wherein:

R_(a) and R_(c) are each, independently, OH, SH, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, amino or substituted amino;

R_(b) is O or S;

X is substituted or unsubstituted C; and wherein X is attached to the 5′-terminal nucleoside. In certain embodiments, X is bound to an atom at the 5′-position of the 5′-terminal nucleoside. In certain such embodiments, the 5′-atom is a carbon and the bond between X and the 5′-carbon of the 5′-terminal nucleoside is a carbon-carbon single bond. In certain embodiments, it is a carbon-carbon double bond. In certain embodiments, it is a carbon-carbon triple bond. In certain embodiments, the 5′-carbon is substituted. In certain embodiments, X is substituted. In certain embodiments, X is unsubstituted.

In certain embodiments, the oligonucleotide optionally comprises a conjugate group and comprises a 5′-stabilized phosphate moiety having the following formula:

wherein:

R_(a) and R_(c) are each, independently, OH, SH, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, amino or substituted amino;

R_(b) is O or S;

X is substituted or unsubstituted C;

Y is selected from C, S, and N. In certain embodiments, Y is substituted or unsubstituted C. The bond between X and Y may be a single-, double-, or triple-bond.

As used herein, “oligonucleotide” means a compound comprising a plurality of linked nucleosides. In certain embodiments, an oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and/or unmodified deoxyribonucleosides (DNA) and/or one or more modified nucleosides.

As used herein “oligonucleoside” means an oligonucleotide in which none of the internucleoside linkages contains a phosphorus atom. As used herein, oligonucleotides include oligonucleosides.

As used herein, “modified oligonucleotide” means an oligonucleotide comprising at least one modified nucleoside and/or at least one modified internucleoside linkage.

As used herein “internucleoside linkage” means a covalent linkage between adjacent nucleosides in an oligonucleotide.

As used herein “naturally occurring internucleoside linkage” means a 3′ to 5′ phosphodiester linkage.

As used herein, “modified internucleoside linkage” means any internucleoside linkage other than a naturally occurring internucleoside linkage.

As used herein, “oligomeric compound” means a polymeric structure comprising two or more sub-structures. In certain embodiments, an oligomeric compound comprises an oligonucleotide. In certain embodiments, an oligomeric compound comprises one or more conjugate groups and/or terminal groups. In certain embodiments, an oligomeric compound consists of an oligonucleotide. Oligomeric compounds also include naturally occurring nucleic acids.

As used herein, “terminal group” means one or more atom attached to either, or both, the 3′ end or the 5′ end of an oligonucleotide. In certain embodiments a terminal group is a conjugate group. In certain embodiments, a terminal group comprises one or more terminal group nucleosides.

As used herein, “conjugate” or “conjugate group” means an atom or group of atoms bound to an oligonucleotide or oligomeric compound. In general, conjugate groups modify one or more properties of the compound to which they are attached, including, but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and/or clearance properties.

As used herein, “conjugate linking group” means any atom or group of atoms used to attach a conjugate to an oligonucleotide or oligomeric compound.

As used herein, “linking group” means any atom or group of atoms used to attach a conjugate to an oligonucleotide or oligomeric compound, or any atom or group of atoms used to attach or connect two different moieties, substrates, linkers, or scaffolds together.

As used herein, “multivalent carbohydrate cluster” means any compound having one or more carbohydrate residues attached to a scaffold or linker group. (see, e.g., Maier et al., “Synthesis of Antisense Oligonucleotides Conjugated to a Multivalent Carbohydrate Cluster for Cellular Targeting,” Bioconjugate Chemistry, 2003, (14): 18-29, which is incorporated herein by reference in its entirety, or Rensen et al., “Design and Synthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic Asiaglycoprotein Receptor,” J. Med. Chem. 2004, (47): 5798-5808, for examples of carbohydrate conjugate clusters).

As used herein, “modified carbohydrate” means any carbohydrate having one or more chemical modifications.

As used herein, “carbohydrate derivative” means any compound which may be synthesized using a carbohydrate as a starting material or intermediate.

As used herein “protecting group” means any compound or protecting group known to those having skill in the art. Non-limiting examples of protecting groups may be found in “Protective Groups in Organic Chemistry”, T. W. Greene, P. G. M. Wuts, ISBN 0-471-62301-6, John Wiley & Sons, Inc, New York, which is incorporated herein by reference in its entirety.

As used herein, “single-stranded” means an oligomeric compound that is not hybridized to its complement and which lacks sufficient self-complementarity to form a stable self-duplex.

As used herein, “antisense compound” means a compound comprising or consisting of an oligonucleotide at least a portion of which is complementary to a target nucleic acid to which it is capable of hybridizing, resulting in at least one antisense activity.

As used herein, “antisense activity” means any detectable and/or measurable change attributable to the hybridization of an antisense compound to its target nucleic acid.

As used herein, “detecting” or “measuring” means that a test or assay for detecting or measuring is performed. Such detection and/or measuring may result in a value of zero. Thus, if a test for detection or measuring results in a finding of no activity (activity of zero), the step of detecting or measuring the activity has nevertheless been performed.

As used herein, “detectable and/or measurable activity” means a statistically significant activity that is not zero.

As used herein, “essentially unchanged” means little or no change in a particular parameter, particularly relative to another parameter which changes much more. In certain embodiments, a parameter is essentially unchanged when it changes less than 5%. In certain embodiments, a parameter is essentially unchanged if it changes less than two-fold while another parameter changes at least ten-fold. For example, in certain embodiments, an antisense activity is a change in the amount of a target nucleic acid. In certain such embodiments, the amount of a non-target nucleic acid is essentially unchanged if it changes much less than the target nucleic acid does, but the change need not be zero.

As used herein, “expression” means the process by which a gene ultimately results in a protein. Expression includes, but is not limited to, transcription, post-transcriptional modification (e.g., splicing, polyadenylation, addition of 5′-cap), and translation.

As used herein, “target nucleic acid” means a nucleic acid molecule to which an antisense compound hybridizes.

As used herein, “targeting” or “targeted to” means the association of an antisense compound to a particular target nucleic acid molecule or a particular region of a target nucleic acid molecule. An antisense compound targets a target nucleic acid if it is sufficiently complementary to the target nucleic acid to allow hybridization under physiological conditions.

As used herein, “nucleobase complementarity” or “complementarity” when in reference to nucleobases means a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In certain embodiments, complementary nucleobase means a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair. Nucleobases comprising certain modifications may maintain the ability to pair with a counterpart nucleobase and thus, are still capable of nucleobase complementarity.

As used herein, “non-complementary” in reference to nucleobases means a pair of nucleobases that do not form hydrogen bonds with one another.

As used herein, “complementary” in reference to oligomeric compounds (e.g., linked nucleosides, oligonucleotides, or nucleic acids) means the capacity of such oligomeric compounds or regions thereof to hybridize to another oligomeric compound or region thereof through nucleobase complementarity. Complementary oligomeric compounds need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated. In certain embodiments, complementary oligomeric compounds or regions are complementary at 70% of the nucleobases (70% complementary). In certain embodiments, complementary oligomeric compounds or regions are 80% complementary. In certain embodiments, complementary oligomeric compounds or regions are 90% complementary. In certain embodiments, complementary oligomeric compounds or regions are 95% complementary. In certain embodiments, complementary oligomeric compounds or regions are 100% complementary.

As used herein, “mismatch” means a nucleobase of a first oligomeric compound that is not capable of pairing with a nucleobase at a corresponding position of a second oligomeric compound, when the first and second oligomeric compound are aligned. Either or both of the first and second oligomeric compounds may be oligonucleotides.

As used herein, “hybridization” means the pairing of complementary oligomeric compounds (e.g., an antisense compound and its target nucleic acid). While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.

As used herein, “specifically hybridizes” means the ability of an oligomeric compound to hybridize to one nucleic acid site with greater affinity than it hybridizes to another nucleic acid site.

As used herein, “fully complementary” in reference to an oligonucleotide or portion thereof means that each nucleobase of the oligonucleotide or portion thereof is capable of pairing with a nucleobase of a complementary nucleic acid or contiguous portion thereof. Thus, a fully complementary region comprises no mismatches or unhybridized nucleobases in either strand.

As used herein, “percent complementarity” means the percentage of nucleobases of an oligomeric compound that are complementary to an equal-length portion of a target nucleic acid. Percent complementarity is calculated by dividing the number of nucleobases of the oligomeric compound that are complementary to nucleobases at corresponding positions in the target nucleic acid by the total length of the oligomeric compound.

As used herein, “percent identity” means the number of nucleobases in a first nucleic acid that are the same type (independent of chemical modification) as nucleobases at corresponding positions in a second nucleic acid, divided by the total number of nucleobases in the first nucleic acid.

As used herein, “modulation” means a change of amount or quality of a molecule, function, or activity when compared to the amount or quality of a molecule, function, or activity prior to modulation. For example, modulation includes the change, either an increase (stimulation or induction) or a decrease (inhibition or reduction) in gene expression. As a further example, modulation of expression can include a change in splice site selection of pre-mRNA processing, resulting in a change in the absolute or relative amount of a particular splice-variant compared to the amount in the absence of modulation.

As used herein, “motif” means a pattern of chemical modifications in an oligonucleotide or a region thereof. Motifs may be defined by modifications at certain nucleosides and/or at certain linking groups of an oligonucleotide.

As used herein, “nucleoside motif” means a pattern of nucleoside modifications in an oligonucleotide or a region thereof. The linkages of such an oligonucleotide may be modified or unmodified. Unless otherwise indicated, motifs herein describing only nucleosides are intended to be nucleoside motifs. Thus, in such instances, the linkages are not limited.

As used herein, “sugar motif” means a pattern of sugar modifications in an oligonucleotide or a region thereof.

As used herein, “linkage motif” means a pattern of linkage modifications in an oligonucleotide or region thereof. The nucleosides of such an oligonucleotide may be modified or unmodified. Unless otherwise indicated, motifs herein describing only linkages are intended to be linkage motifs. Thus, in such instances, the nucleosides are not limited.

As used herein, “nucleobase modification motif” means a pattern of modifications to nucleobases along an oligonucleotide. Unless otherwise indicated, a nucleobase modification motif is independent of the nucleobase sequence.

As used herein, “sequence motif” means a pattern of nucleobases arranged along an oligonucleotide or portion thereof. Unless otherwise indicated, a sequence motif is independent of chemical modifications and thus may have any combination of chemical modifications, including no chemical modifications.

As used herein, “type of modification” in reference to a nucleoside or a nucleoside of a “type” means the chemical modification of a nucleoside and includes modified and unmodified nucleosides. Accordingly, unless otherwise indicated, a “nucleoside having a modification of a first type” may be an unmodified nucleoside.

As used herein, “differently modified” mean chemical modifications or chemical substituents that are different from one another, including absence of modifications. Thus, for example, a MOE nucleoside and an unmodified DNA nucleoside are “differently modified,” even though the DNA nucleoside is unmodified. Likewise, DNA and RNA are “differently modified,” even though both are naturally-occurring unmodified nucleosides. Nucleosides that are the same but for comprising different nucleobases are not differently modified. For example, a nucleoside comprising a 2′-OMe modified sugar and an unmodified adenine nucleobase and a nucleoside comprising a 2′-OMe modified sugar and an unmodified thymine nucleobase are not differently modified.

As used herein, “the same type of modifications” refers to modifications that are the same as one another, including absence of modifications. Thus, for example, two unmodified DNA nucleosides have “the same type of modification,” even though the DNA nucleoside is unmodified. Such nucleosides having the same type modification may comprise different nucleobases.

As used herein, “separate regions” means portions of an oligonucleotide wherein the chemical modifications or the motif of chemical modifications of any neighboring portions include at least one difference to allow the separate regions to be distinguished from one another.

As used herein, “pharmaceutically acceptable carrier or diluent” means any substance suitable for use in administering to an animal. In certain embodiments, a pharmaceutically acceptable carrier or diluent is sterile saline. In certain embodiments, such sterile saline is pharmaceutical grade saline.

As used herein, “substituent” and “substituent group,” means an atom or group that replaces the atom or group of a named parent compound. For example a substituent of a modified nucleoside is any atom or group that differs from the atom or group found in a naturally occurring nucleoside (e.g., a modified 2′-substitutent is any atom or group at the 2′-position of a nucleoside other than H or OH). Substituent groups can be protected or unprotected. In certain embodiments, compounds of the present disclosure have substituents at one or at more than one position of the parent compound. Substituents may also be further substituted with other substituent groups and may be attached directly or via a linking group such as an alkyl or hydrocarbyl group to a parent compound.

Likewise, as used herein, “substituent” in reference to a chemical functional group means an atom or group of atoms that differs from the atom or a group of atoms normally present in the named functional group. In certain embodiments, a substituent replaces a hydrogen atom of the functional group (e.g., in certain embodiments, the substituent of a substituted methyl group is an atom or group other than hydrogen which replaces one of the hydrogen atoms of an unsubstituted methyl group). Unless otherwise indicated, groups amenable for use as substituents include without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (—C(O)R_(aa)), carboxyl (—C(O)O—R_(aa)), aliphatic groups, alicyclic groups, alkoxy, substituted oxy (—O—R_(aa)), aryl, aralkyl, heterocyclic radical, heteroaryl, heteroarylalkyl, amino (—N(R_(bb))(R_(cc))), imino (═NR_(bb)), amido (—C(O)N(R_(bb))(R_(cc)) or —N(R_(bb))C(O)R_(aa)), azido (—N₃), nitro (—NO₂), cyano (—CN), carbamido (—OC(O)N(R_(bb))(R_(cc)) or —N(R_(bb))C(O)OR_(aa)), ureido (—N(R_(bb))C(O)N(R_(bb))(R_(cc))), thioureido (—N(R_(bb))C(S)N(R_(bb))—(R_(cc))), guanidinyl (—N(R_(bb))C(═NR_(bb))N(R_(bb))(R_(cc))), amidinyl (—C(═NR_(bb))N(R_(bb))(R_(cc)) or —N(R_(bb))C(═NR_(bb))(R_(aa))), thiol (—SR_(bb)), sulfinyl (—S(O)R_(bb)), sulfonyl (—S(O)₂R_(bb)) and sulfonamidyl (—S(O)₂N(R_(bb))(R_(cc)) or —N(R_(bb))S—(O)₂R_(bb)). Wherein each R_(aa), R_(bb) and R_(cc) is, independently, H, an optionally linked chemical functional group or a further substituent group with a preferred list including without limitation, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic and heteroarylalkyl. Selected substituents within the compounds described herein are present to a recursive degree.

As used herein, “alkyl,” as used herein, means a saturated straight or branched hydrocarbon radical containing up to twenty four carbon atoms. Examples of alkyl groups include without limitation, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like. Alkyl groups typically include from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms (C₁-C₁₂ alkyl) with from 1 to about 6 carbon atoms being more preferred.

As used herein, “alkenyl,” means a straight or branched hydrocarbon chain radical containing up to twenty four carbon atoms and having at least one carbon-carbon double bond. Examples of alkenyl groups include without limitation, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene and the like. Alkenyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkenyl groups as used herein may optionally include one or more further substituent groups.

As used herein, “alkynyl,” means a straight or branched hydrocarbon radical containing up to twenty four carbon atoms and having at least one carbon-carbon triple bond. Examples of alkynyl groups include, without limitation, ethynyl, 1-propynyl, 1-butynyl, and the like. Alkynyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkynyl groups as used herein may optionally include one or more further substituent groups.

As used herein, “acyl,” means a radical formed by removal of a hydroxyl group from an organic acid and has the general Formula —C(O)—X where X is typically aliphatic, alicyclic or aromatic. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphatic phosphates and the like. Acyl groups as used herein may optionally include further substituent groups.

As used herein, “alicyclic” means a cyclic ring system wherein the ring is aliphatic. The ring system can comprise one or more rings wherein at least one ring is aliphatic. Preferred alicyclics include rings having from about 5 to about 9 carbon atoms in the ring. Alicyclic as used herein may optionally include further substituent groups.

As used herein, “aliphatic” means a straight or branched hydrocarbon radical containing up to twenty four carbon atoms wherein the saturation between any two carbon atoms is a single, double or triple bond. An aliphatic group preferably contains from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms with from 1 to about 6 carbon atoms being more preferred. The straight or branched chain of an aliphatic group may be interrupted with one or more heteroatoms that include nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groups interrupted by heteroatoms include without limitation, polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines. Aliphatic groups as used herein may optionally include further substituent groups.

As used herein, “alkoxy” means a radical formed between an alkyl group and an oxygen atom wherein the oxygen atom is used to attach the alkoxy group to a parent molecule. Examples of alkoxy groups include without limitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groups as used herein may optionally include further substituent groups.

As used herein, “aminoalkyl” means an amino substituted C₁-C₁₂ alkyl radical. The alkyl portion of the radical forms a covalent bond with a parent molecule. The amino group can be located at any position and the aminoalkyl group can be substituted with a further substituent group at the alkyl and/or amino portions.

As used herein, “aralkyl” and “arylalkyl” mean an aromatic group that is covalently linked to a C₁-C₁₂ alkyl radical. The alkyl radical portion of the resulting aralkyl (or arylalkyl) group forms a covalent bond with a parent molecule. Examples include without limitation, benzyl, phenethyl and the like. Aralkyl groups as used herein may optionally include further substituent groups attached to the alkyl, the aryl or both groups that form the radical group.

As used herein, “aryl” and “aromatic” mean a mono- or polycyclic carbocyclic ring system radicals having one or more aromatic rings. Examples of aryl groups include without limitation, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. Preferred aryl ring systems have from about 5 to about 20 carbon atoms in one or more rings. Aryl groups as used herein may optionally include further substituent groups.

As used herein, “halo” and “halogen,” mean an atom selected from fluorine, chlorine, bromine and iodine.

As used herein, “heteroaryl,” and “heteroaromatic,” mean a radical comprising a mono- or polycyclic aromatic ring, ring system or fused ring system wherein at least one of the rings is aromatic and includes one or more heteroatoms. Heteroaryl is also meant to include fused ring systems including systems where one or more of the fused rings contain no heteroatoms. Heteroaryl groups typically include one ring atom selected from sulfur, nitrogen or oxygen. Examples of heteroaryl groups include without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl and the like. Heteroaryl radicals can be attached to a parent molecule directly or through a linking moiety such as an aliphatic group or hetero atom. Heteroaryl groups as used herein may optionally include further substituent groups.

As used herein, “oligonucleotide-conjugate complex” means an oligonucleotide comprising one or more conjugate groups attached to one or more nucleosides on the oligonucleotide.

As used herein, unless otherwise indicated or modified, the term “double-stranded” refers to two separate oligomeric compounds that are hybridized to one another. Such double stranded compounds may have one or more or non-hybridizing nucleosides at one or both ends of one or both strands (overhangs) and/or one or more internal non-hybridizing nucleosides (mismatches) provided there is sufficient complementarity to maintain hybridization under physiologically relevant conditions.

B. CERTAIN COMPOUNDS

In certain embodiments, oligomeric compounds comprising one or more conjugates are described. In certain embodiments, oligomeric compounds comprising an oligonucleotide and a conjugate group and/or terminal group are described. In certain embodiments, oligomeric compounds comprising an oligonucleotide and a conjugate group selectively placed at a nucleoside are described.

a. Certain 5′-Terminal Nucleosides

In certain embodiments, compounds of the present disclosure comprise oligonucleotides comprising a stabilized phosphate moiety at the 5′-terminus. In certain embodiments, such oligonucleotides comprise a 5′ terminal nucleoside having Formula I:

wherein:

T₁ is a phosphorus moiety;

T₂ is an internucleoside linking group linking the nucleoside of Formula I to the remainder of the oligonucleotide;

A has one of the formulas:

Q₁ and Q₂ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy or N(R₃)(R₄);

Q₃ is O, S, N(R₅) or C(R₆)(R₇);

each R₃, R₄, R₅, R₆ and R₇ is, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl or C₁-C₆ alkoxy;

M₃ is O, S, NR₁₄, C(R₁₅)(R₁₆), C(R₁₅)(R₁₆)C(R₁₇)(R₁₈), C(R₁₅)═C(R₁₇), OC(R₁₅)(R₁₆) or OC(R₁₅)(Bx₂);

R₁₄ is H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

R₁₅, R₁₆, R₁₇ and R₁₈ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

one of Bx₁ and Bx₂ is a nucleobase and the other of Bx₁ and Bx₂, if present, is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

J₄, J₅, J₆ and J₇ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

or J₄ forms a bridge with either J₅ or J₇ wherein said bridge comprises from 1 to 3 linked biradical groups selected from O, S, NR₁₉, C(R₂₀)(R₂₁), C(R₂₀)═C(R₂₁), C[═C(R₂₀)(R₂₁)] and C(═O) and the other two of J₅, J₆ and J₇ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

each R₁₉, R₂₀ and R₂₁ is, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

G is H, OH, halogen or O—[C(R₈)(R₉)]_(n)—[(C═O)_(m)—X₁]_(j)—Z, or a conjugate group;

each R₈ and R₉ is, independently, H, halogen, C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

X₁ is O, S or N(E₁);

Z is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or N(E₂)(E₃);

E₁, E₂ and E₃ are each, independently, H, C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

n is from 1 to about 6;

m is 0 or 1;

j is 0 or 1;

each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ₁, N(J₁)(J₂), ═NJ₁, SJ₁, N₃, CN, OC(═X₂)J₁, OC(═X₂)N(J₁)(J₂) and C(═X₂)N(J₁)(J₂);

X₂ is O, S or NJ₃;

each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl; and

when j is 1 then Z is other than halogen or N(E₂)(E₃).

In certain embodiments, oligonucleotides comprise a 5′-terminal nucleoside having Formula II:

wherein:

Bx is a nucleobase;

T₁ is an phosphorus moiety;

T₂ is an internucleoside linking group linking the compound of Formula II to the remainder of the oligonucleotide;

A has one of the formulas:

Q₁ and Q₂ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy or N(R₃)(R₄);

Q₃ is O, S, N(R₅) or C(R₆)(R₇);

each R₃, R₄, R₅, R₆ and R₇ is, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl or C₁-C₆ alkoxy;

G is H, OH, halogen, O—[C(R₈)(R₉)]_(n)—[(C═O)_(m)—X]_(j)—Z or a conjugate group;

each R₈ and R₉ is, independently, H, halogen, C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

X is O, S or N(E₁);

Z is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or N(E₂)(E₃);

E₁, E₂ and E₃ are each, independently, H, C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

n is from 1 to about 6;

m is 0 or 1;

j is 0 or 1;

each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ₁, N(J₁)(J₂), ═NJ₁, SJ₁, N₃, CN, OC(=L)J₁, OC(=L)N(J₁)(J₂) and C(=L)N(J₁)(J₂);

L is O, S or NJ₃;

each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl; and

when j is 1 then Z is other than halogen or N(E₂)(E₃).

In certain embodiments, oligonucleotides comprise a 5′-terminal nucleoside having Formula III:

wherein:

Bx is a nucleobase;

T₁ is a phosphorus moiety;

T₂ is an internucleoside linking group linking the compound of Formula III to the remainder of the oligonucleotide;

A has one of the formulas:

Q₁ and Q₂ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy or N(R₃)(R₄);

Q₃ is O, S, N(R₅) or C(R₆)(R₇);

each R₃, R₄, R₅, R₆ and R₇ is, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl or C₁-C₆ alkoxy;

G is H, OH, halogen, O—[C(R₈)(R₉)]_(n)—[(C═O)_(m)—X]_(j)—Z, or a conjugate group;

each R₈ and R₉ is, independently, H, halogen, C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

X is O, S or N(E₁);

Z is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or N(E₂)(E₃);

E₁, E₂ and E₃ are each, independently, H, C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

n is from 1 to about 6;

m is 0 or 1;

j is 0 or 1;

each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ₁, N(J₁)(J₂), ═NJ₁, SJ₁, N₃, CN, OC(=L)J₁, OC(=L)N(J₁)(J₂) and C(=L)N(J₁)(J₂);

L is O, S or NJ₃;

each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl; and

when j is 1 then Z is other than halogen or N(E₂)(E₃).

In certain embodiments, oligonucleotides comprise a 5′-terminal nucleoside having Formula IV:

In certain embodiments, oligonucleotides are provided comprising a compound having Formula IV wherein Q₁ and Q₂ are each H. In certain embodiments, oligonucleotide are provided comprising a compound having Formula IV wherein G is O(CH₂)₂OCH₃.

In certain embodiments, oligonucleotides comprise a 5′-terminal nucleoside having Formula IV:

wherein:

T₁ is an optionally protected phosphorus moiety;

T₂ is an internucleoside linking group linking the compound of Formula I to the remainder of the oligomeric compound;

Z₁C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆ alkyl, substituted C₂-C₆ alkenyl or substituted C₂-C₆ alkynyl;

M₁ is H, OH or OR₁;

M₂ is OH, OR₁ or N(R₁)(R₂);

each R₁ and R₂ is, independently, C₁-C₆ alkyl, substituted C₁-C₆ alkyl;

r is 0 or 1;

each R₃, R₄, R₅, R₆ and R₇ is, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl or C₁-C₆ alkoxy;

M₃ is O, S, NR₁₄, C(R₁₅)(R₁₆), C(R₁₅)(R₁₆)C(R₁₇)(R₁₈), C(R₁₅)═C(R₁₇), OC(R₁₅)(R₁₆) or OC(R₁₅)(Bx₂);

R₁₄ is H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

R₁₅, R₁₆, R₁₇ and R₁₈ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

one of Bx₁ and Bx₂ is a heterocyclic base moiety and the other of Bx₁ and Bx₂, if present, is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

J₄, J₅, J₆ and J₇ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

or J₄ forms a bridge with either J₅ or J₇ wherein said bridge comprises from 1 to 3 linked biradical groups selected from O, S, NR₁₉, C(R₂₀)(R₂₁), C(R₂₀)═C(R₂₁), C[═C(R₂₀)(R₂₁)] and C(═O) and the other two of J₅, J₆ and J₇ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

each R₁₉, R₂₀ and R₂₁ is, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

G is H, OH, halogen, a conjugate group, or O—[C(R₈)(R₉)]_(n)—[(C═O)_(m)—X₁]_(j)—Z;

each R₈ and R₉ is, independently, H, halogen, C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

X₁ is O, S or N(E₁);

Z is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or N(E₂)(E₃);

E₁, E₂ and E₃ are each, independently, H, C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

n is from 1 to about 6;

m is 0 or 1;

j is 0 or 1;

each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ₁, N(J₁)(J₂), ═NJ₁, SJ₁, N₃, CN, OC(═X₂)J₁, OC(═X₂)N(J₁)(J₂) and C(═X₂)N(J₁)(J₂);

X₂ is O, S or NJ₃;

each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl; and

when j is 1 then Z is other than halogen or N(E₂)(E₃).

In certain embodiments, oligonucleotides comprise a 5′-terminal nucleoside having Formula VI:

In certain embodiments, oligonucleotides comprise a nucleoside of Formula I, II, III, IV, V, or VI. In certain such embodiments, the nucleoside of Formula I, II, III, IV, V, or VI is at the 5′-terminus. In certain such embodiments, the remainder of the oligonucleotide comprises one or more modifications. Such modifications may include modified sugar moieties, modified nucleobases and/or modified internucleoside linkages. Certain such modifications which may be incorporated in an oligonucleotide comprising a nucleoside of Formula I, II, III, IV, V, or VI at the 5′-terminus are known in the art.

b. Certain Sugar Moieties

In certain embodiments, compounds of the disclosure comprise one or more modified nucleosides comprising a modified sugar moiety. Such compounds comprising one or more sugar-modified nucleosides may have desirable properties, such as enhanced nuclease stability or increased binding affinity with a target nucleic acid relative to an oligonucleotide comprising only nucleosides comprising naturally occurring sugar moieties. In certain embodiments, modified sugar moieties are substituted sugar moieties. In certain embodiments, modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of substituted sugar moieties.

In certain embodiments, modified sugar moieties are substituted sugar moieties comprising one or more non-bridging sugar substituent, including but not limited to substituents at the 2′ and/or 5′ positions. Examples of sugar substituents suitable for the 2′-position, include, but are not limited to: 2′-F, 2′-OCH₃ (“OMe” or “O-methyl”), and 2′-O(CH₂)₂OCH₃ (“MOE”). In certain embodiments, sugar substituents at the 2′ position is selected from allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, O—C₁-C₁₀ substituted alkyl; OCF₃, O(CH₂)₂SCH₃, O(CH₂)₂—O—N(Rm)(Rn), and O—CH₂—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C₁-C₁₀ alkyl. Examples of sugar substituents at the 5′-position, include, but are not limited to: 5′-methyl (R or S); 5′-vinyl, and 5′-methoxy. In certain embodiments, substituted sugars comprise more than one non-bridging sugar substituent, for example, 2′-F-5′-methyl sugar moieties (see, e.g., PCT International Application WO 2008/101157, for additional 5′,2′-bis substituted sugar moieties and nucleosides).

Nucleosides comprising 2′-substituted sugar moieties are referred to as 2′-substituted nucleosides. In certain embodiments, a 2′-substituted nucleoside comprises a 2′-substituent group selected from halo, allyl, amino, azido, SH, CN, OCN, CF₃, OCF₃, O, S, or N(R_(m))-alkyl; O, S, or N(R_(m))-alkenyl; O, S or N(R_(m))-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)) or O—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is, independently, H, an amino protecting group or substituted or unsubstituted C₁-C₁₀ alkyl. These 2′-substituent groups can be further substituted with one or more substituent groups independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO₂), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

In certain embodiments, a 2′-substituted nucleoside comprises a 2′-substituent group selected from F, NH₂, N₃, OCF₃, O—CH₃, O(CH₂)₃NH₂, CH₂—CH═CH₂, O—CH₂—CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)), O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N-substituted acetamide (O—CH₂—C(═O)—N(R_(m))(R_(n)) where each R_(m) and R_(n) is, independently, H, an amino protecting group or substituted or unsubstituted C₁-C₁₀ alkyl.

In certain embodiments, a 2′-substituted nucleoside comprises a sugar moiety comprising a 2′-substituent group selected from F, OCF₃, O—CH₃, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(CH₃)₂, —O(CH₂)₂O(CH₂)₂N(CH₃)₂, and O—CH₂—C(═O)—N(H)CH₃.

In certain embodiments, a 2′-substituted nucleoside comprises a sugar moiety comprising a 2′-substituent group selected from F, O—CH₃, and OCH₂CH₂OCH₃.

Certain modified sugar moieties comprise a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety comprises a bridge between the 4′ and the 2′ furanose ring atoms. Examples of such 4′ to 2′ sugar substituents, include, but are not limited to: —[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—, —C(R_(a)R_(b))—N(R)—O— or, —C(R_(a)R_(b))—O—N(R)—; 4′- CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′, 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2; 4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ (cEt) and 4′-CH(CH₂OCH₃)—O-2′, and analogs thereof (see, e.g., U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-C(CH₃)(CH₃)—O-2′ and analogs thereof, (see, e.g., WO2009/006478, published Jan. 8, 2009); 4′-CH₂—N(OCH₃)-2′ and analogs thereof (see, e.g., WO2008/150729, published Dec. 11, 2008); 4′-CH₂—O—N(CH₃)-2′ (see, e.g., US2004/0171570, published Sep. 2, 2004); 4′-CH₂—O—N(R)-2′, and 4′-CH₂—N(R)—O-2′-, wherein each R is, independently, H, a protecting group, or C₁-C₁₂ alkyl; 4′-CH₂—N(R)—O-2′, wherein R is H, C₁-C₁₂ alkyl, or a protecting group (see, U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4′-CH₂—C(H)(CH₃)-2′ (see, e.g., Chattopadhyaya, et al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH₂—C(═CH₂)-2′ and analogs thereof (see, published PCT International Application WO 2008/154401, published on Dec. 8, 2008).

In certain embodiments, such 4′ to 2′ bridges independently comprise from 1 to 4 linked groups independently selected from —[C(R_(a))(R_(b))]_(n)—, —C(R_(a))═C(R_(b))—, —C(R_(a))═N—, —C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—, —Si(R_(a))₂—, —S(═O)_(x)—, and —N(R_(a))—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical, substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃, COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), or sulfoxy (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl (C(═O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl, or a protecting group.

Nucleosides comprising bicyclic sugar moieties are referred to as bicyclic nucleosides or BNAs. Bicyclic nucleosides include, but are not limited to, (A) α-L-Methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-Methyleneoxy (4′-CH₂—O-2′) BNA (also referred to as locked nucleic acid or LNA), (C) Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) Aminooxy (4′-CH₂—O—N(R)-2′) BNA, (E) Oxyamino (4′-CH₂—N(R)—O-2′) BNA, (F) Methyl(methyleneoxy) (4′-CH(CH₃)—O-2′) BNA (also referred to as constrained ethyl or cEt), (G) methylene-thio (4′-CH₂—S-2′) BNA, (H) methylene-amino (4′-CH2-N(R)-2′) BNA, (I) methyl carbocyclic (4′-CH₂—CH(CH₃)-2′) BNA, and (J) propylene carbocyclic (4′-(CH₂)₃-2′) BNA as depicted below.

wherein Bx is a nucleobase moiety and R is, independently, H, a protecting group, or C₁-C₁₂ alkyl.

Additional bicyclic sugar moieties are known in the art, for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 129(26) 8362-8379 (Jul. 4, 2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461, and 7,399,845; WO 2004/106356, WO 1994/14226, WO 2005/021570, and WO 2007/134181; U.S. Patent Publication Nos. US2004/0171570, US2007/0287831, and US2008/0039618; U.S. patent Ser. Nos. 12/129,154, 60/989,574, 61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787, and 61/099,844; and PCT International Applications Nos. PCT/US2008/064591, PCT/US2008/066154, and PCT/US2008/068922.

In certain embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration. For example, a nucleoside comprising a 4′-2′ methylene-oxy bridge, may be in the α-L configuration or in the β-D configuration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′) bicyclic nucleosides have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

In certain embodiments, substituted sugar moieties comprise one or more non-bridging sugar substituent and one or more bridging sugar substituent (e.g., 5′-substituted and 4′-2′ bridged sugars). (see, PCT International Application WO 2007/134181, published on Nov. 22, 2007, wherein LNA is substituted with, for example, a 5′-methyl or a 5′-vinyl group).

In certain embodiments, modified sugar moieties are sugar surrogates. In certain such embodiments, the oxygen atom of the naturally occurring sugar is substituted, e.g., with a sulfur, carbon or nitrogen atom. In certain such embodiments, such modified sugar moiety also comprises bridging and/or non-bridging substituents as described above. For example, certain sugar surrogates comprise a 4′-sulfur atom and a substitution at the 2′-position (see, e.g., published U.S. Patent Application US2005/0130923, published on Jun. 16, 2005) and/or the 5′ position. By way of additional example, carbocyclic bicyclic nucleosides having a 4′-2′ bridge have been described (see, e.g., Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740).

In certain embodiments, sugar surrogates comprise rings having other than 5-atoms. For example, in certain embodiments, a sugar surrogate comprises a six-membered tetrahydropyran. Such tetrahydropyrans may be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid (HNA), anitol nucleic acid (ANA), mannitol nucleic acid (MNA) (see Leumann, C J. Bioorg. & Med. Chem. (2002) 10:841-854), fluoro HNA (F-HNA), and those compounds having Formula VI:

wherein independently for each of said at least one tetrahydropyran nucleoside analog of Formula VII:

Bx is a nucleobase moiety;

T₃ and T₄ are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of T₃ and T₄ is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugate group, or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, or substituted C₂-C₆ alkynyl; and

each of R₁ and R₂ is independently selected from among: hydrogen, halogen, substituted or unsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂, and CN, wherein X is O, S or NJ₁, and each J₁, J₂, and J₃ is, independently, H or C₁-C₆ alkyl.

In certain embodiments, the modified THP nucleosides of Formula VI are provided wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other than H. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is methyl. In certain embodiments, THP nucleosides of Formula VI are provided wherein one of R₁ and R₂ is F. In certain embodiments, R₁ is fluoro and R₂ is H, R₁ is methoxy and R₂ is H, and R₁ is methoxyethoxy and R₂ is H.

Many other bicyclo and tricyclo sugar surrogate ring systems are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds (see, e.g., review article: Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).

Combinations of modifications are also provided without limitation, such as 2′-F-5′-methyl substituted nucleosides (see PCT International Application WO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5′,2′-bis substituted nucleosides) and replacement of the ribosyl ring oxygen atom with S and further substitution at the 2′-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5′-substitution of a bicyclic nucleic acid (see PCT International Application WO 2007/134181, published on Nov. 22, 2007 wherein a 4′-CH₂—O-2′ bicyclic nucleoside is further substituted at the 5′ position with a 5′-methyl or a 5′-vinyl group). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (see, e.g., Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

In certain embodiments, the present disclosure provides oligonucleotides comprising modified nucleosides. Those modified nucleotides may include modified sugars, modified nucleobases, and/or modified linkages. The specific modifications are selected such that the resulting oligonucleotides possess desirable characteristics. In certain embodiments, oligonucleotides comprise one or more RNA-like nucleosides. In certain embodiments, oligonucleotides comprise one or more DNA-like nucleotides.

c. Certain Nucleobases

In certain embodiments, nucleosides of the present disclosure comprise one or more unmodified nucleobases. In certain embodiments, nucleosides of the present disclosure comprise one or more modified nucleobases.

In certain embodiments, modified nucleobases are selected from: universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288.

Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include without limitation, U.S. Pat. Nos. 3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985; 5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

d. Certain Internucleoside Linkages

In certain embodiments, the present disclosure provides oligonucleotides comprising linked nucleosides. In such embodiments, nucleosides may be linked together using any internucleoside linkage. The two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters (P═O), phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (P═S). Representative non-phosphorus containing internucleoside linking groups include, but are not limited to, methylenemethylimino (—CH₂—N(CH₃)—O—CH₂—), thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—); siloxane (—O—Si(H)₂—O—); and N,N′-dimethylhydrazine (—CH₂—N(CH₃)—N(CH₃)—). Modified linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide. In certain embodiments, internucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing internucleoside linkages are well known to those skilled in the art.

The oligonucleotides described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), □ or □ such as for sugar anomers, or as (D) or (L) such as for amino acids etc. Included in the antisense compounds provided herein are all such possible isomers, as well as their racemic and optically pure forms.

Neutral internucleoside linkages include without limitation, phosphotriesters, methylphosphonates, MMI (3′-CH₂—N(CH₃)—O-52 amide-3 (3′-CH₂—C(═O)—N(H)-5′), amide-4 (3′-CH₂—N(H)—C(═O)-5′), formacetal (3′-O—CH₂—O-5′), and thioformacetal (Y—S—CH₂—O-5′). Further neutral internucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral internucleoside linkages include nonionic linkages comprising mixed N, O, S and CH₂ component parts.

e. Certain Motifs

i. Certain Sugar Motifs

In certain embodiments, oligonucleotides comprise one or more type of modified sugar moieties and/or naturally occurring sugar moieties arranged along an oligonucleotide or region thereof in a defined pattern or sugar modification motif. Such motifs may include any of the sugar modifications discussed herein and/or other known sugar modifications.

In certain embodiments, the oligonucleotides comprise or consist of a region having uniform sugar modifications. In certain such embodiments, each nucleoside of the region comprises the same RNA-like sugar modification. In certain embodiments, each nucleoside of the region is a 2′-F nucleoside. In certain embodiments, each nucleoside of the region is a 2′-OMe nucleoside. In certain embodiments, each nucleoside of the region is a 2′-MOE nucleoside. In certain embodiments, each nucleoside of the region is a cEt nucleoside. In certain embodiments, each nucleoside of the region is an LNA nucleoside. In certain embodiments, the uniform region constitutes all or essentially all of the oligonucleotide. In certain embodiments, the region constitutes the entire oligonucleotide except for 1-4 terminal nucleosides.

In certain embodiments, oligonucleotides of the present disclosure comprise one or more regions of alternating sugar modifications, wherein the nucleosides alternate between nucleotides having a sugar modification of a first type and nucleotides having a sugar modification of a second type. In certain embodiments, nucleosides of both types are RNA-like nucleosides. In certain embodiments the alternating nucleosides are selected from: 2′-OMe, 2′-F, 2′-MOE, LNA, and cEt. In certain embodiments, the alternating modifications are 2′-F and 2′-OMe. Such regions may be contiguous or may be interrupted by differently modified nucleosides or conjugated nucleosides.

In certain embodiments, the alternating region of alternating modifications each consist of a single nucleoside (i.e., the pattern is (AB)_(x)A_(y) wherein A is a nucleoside having a sugar modification of a first type and B is a nucleoside having a sugar modification of a second type; x is 1-20 and y is 0 or 1). In certain embodiments, one or more alternating regions in an alternating motif includes more than a single nucleoside of a type. For example, oligonucleotides of the present disclosure may include one or more regions of any of the following nucleoside motifs:

AABBAA;

ABBABB;

AABAAB;

ABBABAABB;

ABABAA;

AABABAB;

ABABAA;

ABBAABBABABAA;

BABBAABBABABAA; or

ABABBAABBABABAA;

wherein A is a nucleoside of a first type and B is a nucleoside of a second type. In certain embodiments, A and B are each selected from 2′-F, 2′-OMe, BNA, and MOE.

In certain embodiments, oligonucleotides having such an alternating motif also comprise a 5′ terminal nucleoside of formula I, II, III, IV, V, or VI.

In certain embodiments, oligonucleotides of the present disclosure comprise a region having a 2-2-3 motif. Such regions comprises the following motif: -(A)₂-(B)_(x)-(A)₂-(C)_(y)-(A)₃

wherein: A is a first type of modified nucleoside;

B and C, are nucleosides that are differently modified than A, however, B and C may have the same or different modifications as one another;

x and y are from 1 to 15.

In certain embodiments, A is a 2′-OMe modified nucleoside. In certain embodiments, B and C are both 2′-F modified nucleosides. In certain embodiments, A is a 2′-OMe modified nucleoside and B and C are both 2′-F modified nucleosides.

It is to be understood, that certain of the above described motifs and modifications may be combined. Since a motif may comprises only a few nucleosides, a particular oligonucleotide may comprise two or more motifs. By way of non-limiting example, in certain embodiments, oligonucleotides may have nucleoside motifs as described in the table below. In the table below, the term “None” indicates that a particular feature is not present in the oligonucleotide. For example, “None” in the column labeled “5′ motif/modification” indicates that the 5′ end of the oligonucleotide comprises the first nucleoside of the central motif

Central 5′ motif/modification Motif 3′-motif Compound of Formula I, II, III, IV, V, or VI Alternating 2 MOE nucleosides Compound of Formula I, II, III, IV, V, or VI 2-2-3 motif 2 MOE nucleosides Compound of Formula I, II, III, IV, V, or VI Uniform 2 MOE nucleosides Compound of Formula I, II, III, IV, V, or VI Alternating 2 MOE nucleosides Compound of Formula I, II, III, IV, V, or VI Alternating 2 MOE A's Compound of Formula I, II, III, IV, V, or VI 2-2-3 motif 2 MOE A's Compound of Formula I, II, III, IV, V, or VI Uniform 2 MOE A's Compound of Formula I, II, III, IV, V, or VI Alternating 2 MOE U's Compound of Formula I, II, III, IV, V, or VI 2-2-3 motif 2 MOE U's Compound of Formula I, II, III, IV, V, or VI Uniform 2 MOE U's Compound of Formula I, II, III, IV, V, or VI Alternating 2 MOE nucleosides Compound of Formula I, II, III, IV, V, or VI 2-2-3 motif 2 MOE nucleosides Compound of Formula I, II, III, IV, V, or VI Uniform 2 MOE nucleosides

In certain embodiments, oligonucleosides have the following sugar motif: 5′-(Q)-(E)_(w)-(A)₂-(B)_(x)-(A)₂-(C)_(y)-(A)₃-(D)_(z) wherein:

Q is a nucleoside comprising a stabilized phosphate moiety. In certain embodiments, Q is a nucleoside having Formula I, II, III, IV, V, or VI;

A is a first type of modified nucleoside;

B, C, D, and E are nucleosides that are differently modified than A, however, B, C, D, and E may have the same or different modifications as one another;

w and z are from 0 to 15;

x and y are from 1 to 15.

In certain embodiments, the sum of w, x, and y is 5-25.

In certain embodiments, oligonucleosides have the following sugar motif: 5′-(Q)-(AB)_(x)A_(y)-(D)_(z) wherein:

Q is a nucleoside comprising a stabilized phosphate moiety. In certain embodiments, Q is a nucleoside having Formula I, II, III, IV, V, or VI;

A is a first type of modified nucleoside;

B is a second type of modified nucleoside;

D is a modified nucleoside comprising a modification different from the nucleoside adjacent to it. Thus, if y is 0, then D must be differently modified than B and if y is 1, then D must be differently modified than A. In certain embodiments, D differs from both A and B.

X is 5-15;

Y is 0 or 1;

Z is 0-4.

In certain embodiments, oligonucleosides have the following sugar motif: 5′-(Q)-(A)_(x)-(D)_(z) wherein:

Q is a nucleoside comprising a stabilized phosphate moiety. In certain embodiments, Q is a nucleoside having Formula I, II, III, IV, V, or VI;

A is a first type of modified nucleoside;

D is a modified nucleoside comprising a modification different from A.

X is 11-30;

Z is 0-4.

In certain embodiments A, B, C, and D in the above motifs are selected from: 2′-F, 2′-MOE, LNA, and cEt. In certain embodiments, D represents terminal nucleosides. In certain embodiments, such terminal nucleosides are not designed to hybridize to the target nucleic acid (though one or more might hybridize by chance). In certain embodiments, the nucleobase of each D nucleoside is adenine, regardless of the identity of the nucleobase at the corresponding position of the target nucleic acid. In certain embodiments the nucleobase of each D nucleoside is thymine.

ii. Certain Internucleoside Linkage Motifs

In certain embodiments, oligonucleotides comprise modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif. In certain embodiments, oligonucleotides comprise a region having an alternating internucleoside linkage motif. In certain embodiments, oligonucleotides of the present disclosure comprise a region of uniformly modified internucleoside linkages. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphorothioate.

In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least one 12 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3′ end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3′ end of the oligonucleotide.

Oligonucleotides having any of the various sugar motifs described herein, may have any linkage motif. For example, the oligonucleotides, including but not limited to those described above, may have a linkage motif selected from non-limiting the table below:

5′ most linkage Central region 3′-region PS Alternating PO/PS 6 PS PS Alternating PO/PS 7 PS PS Alternating PO/PS 8 PS

iii. Certain Nucleobase Modification Motifs

In certain embodiments, oligonucleotides comprise chemical modifications to nucleobases arranged along the oligonucleotide or region thereof in a defined pattern or nucleobases modification motif. In certain such embodiments, nucleobase modifications are arranged in a gapped motif. In certain embodiments, nucleobase modifications are arranged in an alternating motif. In certain embodiments, each nucleobase is modified. In certain embodiments, none of the nucleobases is chemically modified.

In certain embodiments, oligonucleotides comprise a block of modified nucleobases. In certain such embodiments, the block is at the 3′-end of the oligonucleotide. In certain embodiments the block is within 3 nucleotides of the 3′-end of the oligonucleotide. In certain such embodiments, the block is at the 5′-end of the oligonucleotide. In certain embodiments the block is within 3 nucleotides of the 5′-end of the oligonucleotide.

In certain embodiments, nucleobase modifications are a function of the natural base at a particular position of an oligonucleotide. For example, in certain embodiments each purine or each pyrimidine in an oligonucleotide is modified. In certain embodiments, each adenine is modified. In certain embodiments, each guanine is modified. In certain embodiments, each thymine is modified. In certain embodiments, each cytosine is modified. In certain embodiments, each uracil is modified.

In certain embodiments, some, all, or none of the cytosine moieties in an oligonucleotide are 5-methyl cytosine moieties. Herein, 5-methyl cytosine is not a “modified nucleobase.” Accordingly, unless otherwise indicated, unmodified nucleobases include both cytosine residues having a 5-methyl and those lacking a 5 methyl. In certain embodiments, the methylation state of all or some cytosine nucleobases is specified.

In certain embodiments, chemical modifications to nucleobases comprise attachment of certain conjugate groups to nucleobases. In certain embodiments, each purine or each pyrimidine in an oligonucleotide may be optionally modified to comprise a conjugate group.

C. CERTAIN OVERALL LENGTHS

In certain embodiments, the present disclosure provides oligonucleotides of any of a variety of ranges of lengths. In certain embodiments, oligonucleotides consist of X to Y linked nucleosides, where X represents the fewest number of nucleosides in the range and Y represents the largest number of nucleosides in the range. In certain such embodiments, X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X<Y. For example, in certain embodiments, the oligonucleotide may consist of 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8 to 13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, 8 to 20, 8 to 21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to 27, 8 to 28, 8 to 29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to 14, 9 to 15, 9 to 16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to 22, 9 to 23, 9 to 24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to 30, 10 to 11, 10 to 12, 10 to 13, 10 to 14, 10 to 15, 10 to 16, 10 to 17, 10 to 18, 10 to 19, 10 to 20, 10 to 21, 10 to 22, 10 to 23, 10 to 24, 10 to 25, 10 to 26, 10 to 27, 10 to 28, 10 to 29, 10 to 30, 11 to 12, 11 to 13, 11 to 14, 11 to 15, 11 to 16, 11 to 17, 11 to 18, 11 to 19, 11 to 20, 11 to 21, 11 to 22, 11 to 23, 11 to 24, 11 to 25, 11 to 26, 11 to 27, 11 to 28, 11 to 29, 11 to 30, 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linked nucleosides. In embodiments where the number of nucleosides of an oligonucleotide of a compound is limited, whether to a range or to a specific number, the compound may, nonetheless further comprise additional other substituents. For example, an oligonucleotide comprising 8-30 nucleosides excludes oligonucleotides having 31 nucleosides, but, unless otherwise indicated, such an oligonucleotide may further comprise, for example one or more conjugate groups, terminal groups, or other substituents.

Further, where an oligonucleotide is described by an overall length range and by regions having specified lengths, and where the sum of specified lengths of the regions is less than the upper limit of the overall length range, the oligonucleotide may have additional nucleosides, beyond those of the specified regions, provided that the total number of nucleosides does not exceed the upper limit of the overall length range.

D. CERTAIN OLIGONUCLEOTIDES

In certain embodiments, oligonucleotides of the present disclosure are characterized by their sugar motif, internucleoside linkage motif, nucleobase modification motif and overall length. In certain embodiments, such parameters are each independent of one another. Thus, each internucleoside linkage of an oligonucleotide having a gapmer sugar motif may be modified or unmodified and may or may not follow the gapmer modification pattern of the sugar modifications. Thus, the internucleoside linkages within the wing regions of a sugar-gapmer may be the same or different from one another and may be the same or different from the internucleoside linkages of the gap region. Likewise, such sugar-gapmer oligonucleotides may comprise one or more modified nucleobase independent of the gapmer pattern of the sugar modifications. One of skill in the art will appreciate that such motifs may be combined to create a variety of oligonucleotides, such as those provided in the non-limiting table below. As is apparent from the above, non-limiting tables, the lengths of the regions defined by a nucleoside motif and that of a linkage motif need not be the same. To further illustrate, and not to limit in any way, nucleoside motifs and sequence motifs are combined to show five non-limiting examples in the table below. The first column of the table lists nucleosides and linkages by position from N1 (the first nucleoside at the 5′-end) to N20 (the 20^(th) position from the 5′-end). In certain embodiments, oligonucleotides of the present disclosure are longer than 20 nucleosides (the table is merely exemplary). Certain positions in the table recite the nucleoside or linkage “none” indicating that the oligonucleotide has no nucleoside at that position.

Pos A B C D E N1 Formula I, II, Formula I, II, Formula I, II, Formula I, II, Formula I, II, III, IV, V, or III, IV, V, or III, IV, V, or III, IV, V, or III, IV, V, or VI VI VI VI VI L1 PS PS PS PS PO N2 2′-F 2′-F 2′-F 2′-OMe MOE L2 PS PS PS PO PS N3 2′-OMe 2′-F 2′-F 2′-F 2′-F L3 PO PS PS PS PS N4 2′-F 2′-F 2′-F 2′-OMe 2′-F L4 PS PS PS PO PS N5 2′-OMe 2′-F 2′-F 2′-F 2′-OMe L5 PO PS PS PS PO N6 2′-F 2′-OMe 2′-F 2′-OMe 2′-OMe L6 PS PO PS PO PO N7 2′-OMe 2′-OMe 2′-F 2′-F 2′-OMe L7 PO PO PS PS PO N8 2′-F 2′-F 2′-F 2′-OMe 2′-F L8 PS PS PS PO PS N9 2′-OMe 2′-F 2′-F 2′-F 2′-F L9 PO PS PS PS PS N10 2′-F 2′-OMe 2′-F 2′-OMe 2′-OMe L10 PS PO PS PO PO N11 2′-OMe 2′-OMe 2′-F 2′-F 2′OMe L11 PO PO PS PS PO N12 2′-F 2′-F 2′-F 2′-F 2′-F L12 PS PS PS PO PS N13 2′-OMe 2′-F 2′-F 2′-F 2′-F L13 PO PS PS PS PS N14 2′-F 2′-OMe 2′-F 2′-F 2′-F L14 PS PS PS PS PS N15 2′-OMe 2′OMe 2′-F 2′-F 2′-MOE L15 PS PS PS PS PS N16 2′-F 2′OMe 2′-F 2′-F 2′-MOE L16 PS PS PS PS PS N17 2′-OMe 2′-MOE U 2′-F 2′-F 2′-MOE L17 PS PS PS PS None N18 2′-F 2′-MOE U 2′-F 2′-OMe None L18 PS None PS PS None N19 2′-MOE U None 2′-MOE U 2′-MOE A None L19 PS None PS PS None N20 2′-MOE U None 2′-MOE U 2′-MOE A None In the above, non-limiting examples:

Column A represent an oligonucleotide consisting of 20 linked nucleosides, wherein the oligonucleotide comprises: a modified 5′-terminal nucleoside of Formula I, II, III, IV, V, or VI; a region of alternating nucleosides; a region of alternating linkages; two 3′-terminal MOE nucleosides, each of which comprises a uracil base; and a region of six phosphorothioate linkages at the 3′-end.

Column B represents an oligonucleotide consisting of 18 linked nucleosides, wherein the oligonucleotide comprises: a modified 5′-terminal nucleoside of Formula I, II, III, IV, V, or VI; a 2-2-3 motif wherein the modified nucleoside of the 2-2-3 motif are 2′O-Me and the remaining nucleosides are all 2′-F; two 3′-terminal MOE nucleosides, each of which comprises a uracil base; and a region of six phosphorothioate linkages at the 3′-end.

Column C represents an oligonucleotide consisting of 20 linked nucleosides, wherein the oligonucleotide comprises: a modified 5′-terminal nucleoside of Formula I, II, III, IV, V, or VI; a region of uniformly modified 2′-F nucleosides; two 3′-terminal MOE nucleosides, each of which comprises a uracil base; and wherein each internucleoside linkage is a phosphorothioate linkage.

Column D represents an oligonucleotide consisting of 20 linked nucleosides, wherein the oligonucleotide comprises: a modified 5′-terminal nucleoside of Formula I, II, III, IV, V, or VI; a region of alternating 2′-OMe/2′-F nucleosides; a region of uniform 2′F nucleosides; a region of alternating phosphorothioate/phosphodiester linkages; two 3′-terminal MOE nucleosides, each of which comprises an adenine base; and a region of six phosphorothioate linkages at the 3′-end.

Column E represents an oligonucleotide consisting of 17 linked nucleosides, wherein the oligonucleotide comprises: a modified 5′-terminal nucleoside of Formula I, II, III, IV, V, or VI; a 2-2-3 motif wherein the modified nucleoside of the 2-2-3 motif are 2′F and the remaining nucleosides are all 2′-OMe; three 3′-terminal MOE nucleosides.

The above examples are provided solely to illustrate how the described motifs may be used in combination and are not intended to limit the disclosure to the particular combinations or the particular modifications used in illustrating the combinations. Further, specific examples herein, including, but not limited to those in the above table are intended to encompass more generic embodiments. For example, column A in the above table exemplifies a region of alternating 2′-OMe and 2′-F nucleosides. Thus, that same disclosure also exemplifies a region of alternating different 2′-modifications. It also exemplifies a region of alternating 2′-O-alkyl and 2′-halogen nucleosides. It also exemplifies a region of alternating differently modified nucleosides. All of the examples throughout this specification contemplate such generic interpretation.

It is also noted that the lengths of the oligonucleotides, such as those exemplified in the above tables, can be easily manipulated by lengthening or shortening one or more of the described regions, without disrupting the motif.

In certain embodiments, the disclosure provides oligonucleotides wherein the 5′-terminal nucleoside (position 1) is a compound of Formula I, II, III, IV, V, or VI and the position 2 nucleoside comprises a 2′-modification. In certain such embodiments, the 2′-modification of the position 2 nucleoside is selected from halogen, alkyl, and substituted alkyl. In certain embodiments, the 2′-modification of the position 2 nucleoside is selected from 2′-F and 2′-alkyl. In certain embodiments, the 2′-modification of the position 2 nucleoside is 2′-F. In certain embodiments, the 2′-substituted of the position 2 nucleoside is an unmodified OH (as in naturally occurring RNA).

In certain embodiments, the position 3 nucleoside is a modified nucleoside. In certain embodiments, the position 3 nucleoside is a bicyclic nucleoside. In certain embodiments, the position 3 nucleoside comprises a sugar surrogate. In certain such embodiments, the sugar surrogate is a tetrahydropyran. In certain embodiments, the sugar of the position 3 nucleoside is a F-HNA.

In certain embodiments, an antisense compound comprises an oligonucleotide comprising 10 to 30 linked nucleosides wherein the oligonucleotide comprises: a position 1 modified nucleoside of Formula I, II, III, IV, V, or VI; a position 2 nucleoside comprising a sugar moiety which is differently modified compared to the sugar moiety of the position 1 modified nucleoside; and from 1 to 4 3′-terminal group nucleosides each comprising a 2′-modification; and wherein at least the seven 3′-most internucleoside linkages are phosphorothioate linkages.

E. CERTAIN CONJUGATE GROUPS

In certain embodiments, oligonucleotides are modified by attachment of one or more conjugate groups. In general, conjugate groups modify one or more properties of the attached oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, cellular distribution, cellular uptake, charge and clearance. Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional conjugate linking moiety or conjugate linking group to a parent compound such as an oligonucleotide. Conjugate groups includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes. Certain conjugate groups have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).

In certain embodiments at least one conjugate group is selected from the group consisting of, folic acid, gallic acid, vitamins, fatty acids, carbohydrates, optical imaging moieties, targeting moieties, magnetic resonance imaging moieties, hydrophobic moieties, hydrophilic moieties, magnetic resonance imaging moieties, peptides, amino acids, amino acid derivatives, nucleic acids, nucleic acid derivatives, heterocycles, steroids, ionic complexes, and polyionic complexes.

In certain embodiments the dye is selected from an acridine dye, a coumarine dye, a rhodamine dye, a xanthene dye, a cyanine dye, and a pyrene dye, Texas Red, Alexa Fluor® dye, BODIPY® dye, Fluorescein, Oregon Green® dye, and Rhodamine Green™ dye or any other suitable dye known to those having skill in the art.

In certain embodiments the conjugate group is a magnetic resonance imaging moiety, for example a paramagnetic compound. In certain embodiments the conjugate group increases the binding affinity to lipoproteins. In certain embodiments the conjugate group is a hydrophobic compound. In certain embodiments the conjugate group is a hydrophilic compound. In certain embodiments the conjugate group is a peptide, an amino acid, an amino acid derivative, a nucleic acid, a nucleic acid derivative, a heterocycle, a steroid, an ionic complex, a polyionic complex, a cationic complex, an anion complex, or a zwitterion.

In certain embodiments, the conjugate group is a carbohydrate, carbohydrate derivative, modified carbohydrate, multivalent carbohydrate cluster, polysaccharide, modified polysaccharide, or polysaccharide derivative. In certain embodiments, the conjugate group is an amino sugar or a thio sugar. For example, amino sugars may be selected from any number of compounds known in the art, for example glucosamine, sialic acid, α-D-galactosamine, N-Acetylgalactosamine, 2-acetamido-2-deoxy-D-galactopyranose (GalNAc), 2-Amino-3-O—[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose (β-muramic acid), 2-Deoxy-2-methylamino-L-glucopyranose, 4,6-Dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-Deoxy-2-sulfoamino-D-glucopyranose and N-sulfo-D-glucosamine, and N-Glycoloyl-α-neuraminic acid. For example, thio sugars may be selected from the group consisting of 5-Thio-β-D-glucopyranose, Methyl 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside, 4-Thio-β-D-galactopyranose, and ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-gluco-heptopyranoside.

In certain embodiments, the multivalent carbohydrate cluster comprises any compound having one or more carbohydrate residues attached to a scaffold or linker group. The scaffold or linker group may comprise any suitable compound known to those in the art. For example, a scaffold or linker group may comprise glycerol or a glycerol derivative, an amine or amine derivative, an ester or ester derivative, an anhydride or anhydride derivative, an amino acid, amino acid derivative, peptide, peptide derivative, or any other suitable compound known to those having skill in the art. For example, in certain embodiments, the scaffold may have the following structure:

wherein each X″₁, X″₂, and X″₃, comprises a linker group and each Y″₁, Y″₂, and Y″₃, comprises a carbohydrate, modified carbohydrate, or carbohydrate derivative. In certain embodiments, each Y″₁, Y″₂, and Y″₃ is independently selected from the group consisting of mannose, glucose, fructose, galactose, amino sugars, and thio sugars.

In some embodiments the multivalent carbohydrate cluster has one carbohydrate attached to the scaffold. In some embodiments the multivalent carbohydrate cluster has two carbohydrates attached to the scaffold. In some embodiments the multivalent carbohydrate cluster has three carbohydrate attached to the scaffold. In some embodiments the multivalent carbohydrate cluster has four carbohydrate attached to the scaffold. In some embodiments the multivalent carbohydrate cluster has one or more carbohydrates attached to the scaffold.

In certain embodiments, the conjugate group comprises:

wherein L″₄ is a linker group, M′ is a scaffold, L″ is a linker group, n is 0 or an integer between 1 and 10, T′ is selected from the group consisting of a carbohydrate, a modified carbohydrate, and a carbohydrate derivative. For example, when n is >0, the scaffold, M′, comprises more than one linker group L″ and more than one carbohydrate, modified carbohydrate, and carbohydrate derivative, T″. For example, when n=2, the scaffold, M′ comprises two separate linker groups, L″₁ and L″₂, wherein each L″₁ and each L″₂ each independently link to two carbohydrates, modified carbohydrates, and carbohydrate derivatives, T′₁ and T′₂.

In certain embodiments, T′, T′₁, T′₂, and T′₃ comprise carbohydrates, modified carbohydrates, and carbohydrate derivatives. In certain embodiments, one or more of T′, T′₁, T′₂, and T′₃ is independently mannose, galactose, D-mannopyranose, L-Mannopyranose, D-Arabinose, L-Galactose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-Galactose, L-Galactose, 2-acetamido-2-deoxy-D-galactopyranose (GalNAc), α-D-Mannofuranose, β-D-Mannofuranose, α-D-Mannopyranose, β-D-Mannopyranose, α-D-Glucopyranose, β-D-Glucopyranose, α-D-Glucofuranose, β-D-Glucofuranose, α-D-fructofuranose, α-D-fructopyranose, α-D-Galactopyranose, β-D-Galactopyranose, α-D-Galactofuranose, β-D-Galactofuranose, glucosamine, sialic acid, α-D-galactosamine, N-Acetylgalactosamine, 2-Amino-3-O—[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-Deoxy-2-methylamino-L-glucopyranose, 4,6-Dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-Deoxy-2-sulfoamino-D-glucopyranose, N-Glycoloyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, methyl 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside, 4-Thio-β-D-galactopyranose, ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-gluco-heptopyranoside, 2,5-Anhydro-D-allononitrile.

In certain embodiments, L″₁, L″₂, L″₃, L″₄, and L″₅ each independently comprise a linker group. In certain embodiments, one or more of L″₁, L″₂, L″₃, L″₄, and L″₅ is independently a peptide, an ether, polyethylene glycol, alkyl, C₁-C₂₀ alkyl, substituted C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, substituted C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, substituted C₂-C₂₀ alkynyl, C₁-C₂₀ alkoxy, substituted C₁-C₂₀ alkoxy, amino, amido, pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and 6-aminohexanoic acid.

In certain embodiments, the linker group comprises an amine. In certain embodiments the linker group comprises polyethylene glycol. In certain embodiments the linker group comprises an ether. In certain embodiments, the linker group comprises a carbamoyl. In certain embodiments, the linker group comprises a carbamate. In certain embodiments, the linker group comprises a structure as shown below:

wherein m and n are 0 or integers between 1 and 20, and wherein Z_(a) and Z_(b) are each independently a conjugate group, a carbohydrate, a modified carbohydrate, a carbohydrate derivative, H, a peptide, ether, polyethylene glycol, alkyl, C₁-C₂₀ alkyl, substituted C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, substituted C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, substituted C₂-C₂₀ alkynyl, C₁-C₂₀ alkoxy, substituted C₁-C₂₀ alkoxy, amino, amido, pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and 6-aminohexanoic acid.

In certain embodiments, the linker group comprises a structure as shown below:

wherein n is 0 or an integer between 1 and 20, and wherein Z_(a) and Z_(b) are each independently a conjugate group, a carbohydrate, a modified carbohydrate, a carbohydrate derivative, H, a peptide, ether, polyethylene glycol, alkyl, C₁-C₂₀ alkyl, substituted C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, substituted C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, substituted C₂-C₂₀ alkynyl, C₁-C₂₀ alkoxy, substituted C₁-C₂₀ alkoxy, amino, amido, pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and 6-aminohexanoic acid.

In certain embodiments, one or more of the hydroxyl groups of the carbohydrates, modified carbohydrates, and carbohydrate derivatives may be protected by a protecting group. Non-limiting examples of protecting groups may be found in “Protective Groups in Organic Chemistry”, T. W. Greene, P. G. M. Wuts, ISBN 0-471-62301-6, John Wiley & Sons, Inc, New York, which is incorporated herein by reference in its entirety. Examples of protecting groups include any protecting group known to those having skill in the art, including acetates, anhydrides, esters, benzyl, substituted benzyl, and allyl. Non-limiting examples of ester protecting groups include acetate, benzoate, chloroacetate, pivalate, levulinate, and silyl ethers. Non-limiting examples of acetals include benzylidene, isopropylidene, and butane diacetal. Non-limiting examples of silyl ether protecting groups include trimethylsilyl (TMS), t-butyldimethylsilyl (TBDMS), and t-butyldiphenylsilyl TBDPS.

In certain embodiments, the multivalent carbohydrate cluster has the following structure:

In certain embodiments, the multivalent carbohydrate cluster has the following structure:

wherein R_(a), R_(b), and R_(c) are each independently a protecting group, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, amino, amido, and:

In certain embodiments, the multivalent carbohydrate cluster has the following structure:

wherein R_(a), R_(b), and R_(c) are each independently a protecting group, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl, C₁-C₆ alkoxy, substituted C_(r) C₆ alkoxy, amino, amido, and:

In certain embodiments, the multivalent carbohydrate cluster has the following structure:

wherein R_(a), R_(b), and R_(c) are each independently a protecting group, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, amino, and amido, and wherein L″₅ is a linker group, wherein the linker group comprises a peptide, an ether, polyethylene glycol, alkyl, C₁-C₂₀ alkyl, substituted C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, substituted C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, substituted C₂-C₂₀ alkynyl, C₁-C₂₀ alkoxy, substituted C₁-C₂₀, alkoxy, amino, amido, pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and 6-aminohexanoic acid, or any other suitable linker group known to those having skill in the art.

In certain embodiments the conjugate group is a targeting moiety. In certain embodiments, the targeting moiety is selected from the group consisting of an arginine-glycine-aspartate (RGD) peptide, fibronectin, folate, galactose, an apolipoprotein, insulin, transferrin, a fibroblast growth factor (FOF), an epidermal growth factor (EGF) and an antibody.

In certain embodiments the conjugate group is a group that increases lipoprotein binding affinity. In certain embodiments, the conjugate group is a group that decreases lipoprotein binding affinity. In certain embodiments, the conjugate group has a pH value of greater than or equal to 7, and in certain embodiments the conjugate group has a pH value of less than or equal to 7.

In certain embodiments the conjugate group increases the lipophilicity of the oligonucleotide-conjugate complex. In certain embodiments the conjugate group decreases the lipophilicity of the oligonucleotide-conjugate complex. In certain embodiments the conjugate group increases the lipophobicity of the oligonucleotide-conjugate complex. In certain embodiments the conjugate group decreases the lipophobicity of the oligonucleotide-conjugate complex.

In certain embodiments, the conjugate group may modify the plasma protein binding properties of the oligonucleotide-conjugate complex. In certain embodiments, the conjugate group may increase and alter the binding affinity of the oligonucleotide for albumin. In certain embodiments, the conjugate group may increase and alter the binding affinity of the oligonucleotide for glycoprotein. In certain embodiments the conjugate group may increase and alter the binding affinity of the oligonucleotide for globulins. In certain embodiments the conjugate group may increase and alter the binding affinity of the oligonucleotide for α-1 globulins, α-2 globulins, β-globulins, and γ-globulins.

In certain embodiments, conjugate groups increase, decrease, or alter the amount of an oligonucleotide that binds to proteins. In certain embodiments, conjugate groups increase, decrease, or alter the amount of an oligonucleotide that binds to plasma proteins. In certain embodiments, conjugate groups increase the amount of an oligonucleotide that binds to plasma proteins. For example, in certain embodiments, the attachment or association of a conjugate group with an oligonucleotide is expected to result in an oligonucleotide-conjugate complex having increased protein binding affinity. In certain embodiments, conjugate groups decrease the amount of an oligonucleotide that binds to plasma proteins. For example, in certain embodiments, the attachment or association of a conjugate group with an oligonucleotide result in an oligonucleotide-conjugate complex having decreased protein binding affinity.

In certain embodiments, conjugate groups increase, decrease, or alter the amount of an oligonucleotide that binds to high molecular weight proteins. In certain embodiments, conjugate groups increase the proportion of an oligonucleotide that binds to high molecular weight proteins. In certain embodiments, the selective placement of conjugate groups along the oligonucleoside increases the proportion of an oligonucleotide that binds to high molecular weight proteins. For example, in certain embodiments, the attachment or association of a conjugate group with an oligonucleotide results in an oligonucleotide-conjugate complex having increased binding affinity for high molecular weight proteins.

In certain embodiments, conjugate groups increase, decrease, or alters the amount of an oligonucleotide that accumulates in one or more tissues. In certain embodiments, conjugate groups increase, decrease, or alters amount of an oligonucleotide that accumulates in adipose tissue. In certain embodiments, conjugate groups increase, decrease, or alters amount of an oligonucleotide that accumulates in the liver. In certain embodiments, conjugate groups increase, decrease, or alters amount of an oligonucleotide that accumulates in the kidney.

In certain embodiments, conjugate groups increase the amount of an oligonucleotide that accumulates in adipose tissue. In certain embodiments, conjugate groups increase the amount of an oligonucleotide that accumulates in the liver. In certain embodiments, conjugate groups increase the amount of an oligonucleotide that accumulates in the kidney.

In certain embodiments, conjugate groups decrease the amount of an oligonucleotide that accumulates in adipose tissue. In certain embodiments, conjugate groups decrease the amount of an oligonucleotide that accumulates in the liver. In certain embodiments, conjugate groups decrease the amount of an oligonucleotide that accumulates in the kidney.

In certain embodiments, conjugate groups may be covalently attached to an oligonucleotide by any one of a number of linkers known to those having skill in the art. In certain embodiments, a linker may be selected to prevent cleavage by any number of enzymes. In certain embodiments, a linker may be selected that is resistant to hydrolytic cleavage. In certain embodiments, a linker may be selected that is resistant to enzymatic cleavage. In certain embodiments, the cleavage resistant linker is selected from a carbamate or carbamoyl moiety.

In certain embodiments, a conjugate group comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. In certain embodiments, a conjugate group comprises an active drug substance, for example, a taxane, a comptotheca, or an anthracycline.

In certain embodiments, conjugate groups are directly attached to oligonucleotides. In certain embodiments, conjugate groups are attached to oligonucleotides by a conjugate linking group. In certain such embodiments, conjugate linking groups, include, but are not limited to, bifunctional linking moieties such as those known in the art are amenable to the compounds provided herein. Conjugate linking groups are useful for attachment of conjugate groups, such as chemical stabilizing groups, functional groups, reporter groups and other groups to selective sites in a parent compound such as for example an oligonucleotide. In general a bifunctional linking moiety comprises a hydrocarbyl moiety having two functional groups. One of the functional groups is selected to bind to a parent molecule or compound of interest and the other is selected to bind essentially any selected group such as chemical functional group or a conjugate group. In some embodiments, the conjugate linker group comprises a chain structure or an oligomer of repeating units such as ethylene glycol or amino acid units. Examples of functional groups that are routinely used in a bifunctional linking moiety include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In some embodiments, bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double or triple bonds), and the like.

Some nonlimiting examples of conjugate linking moieties include polyethylene glycol (PEG), pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and 6-aminohexanoic acid (AHEX or AHA). Other linking groups include, but are not limited to, substituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl or substituted or unsubstituted C₂-C₁₀ alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

In certain embodiments, the linking group is represented by a compound of formula VIII:

wherein, n is any integer between 1 and 10, R′ comprises a nucleoside, and R″ comprises a conjugate group, or has the formula R″(R_(1a))(R_(1b))(R_(1c)), wherein R_(1a), R_(2a), and R_(3a) are each, independently, a conjugate group, H, halogen, C₁-C₂₄ alkyl, substituted C₁-C₂₄ alkyl, C₁-C₂₄ alkoxy, substituted C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyl, substituted C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, substituted C₂-C₂₄ alkynyl, or where R_(1a) and one of R_(2a) or R_(3a) together form Q′, where Q′ is O.

For example, in certain embodiments, the linking group is attached to a nucleoside as shown below:

In certain embodiments, the linking group comprises a thiodiester, thionocarbamate, siloxane, carbamate, sulfamate, morpholino sulfamide, sulfonamide, sulfide, sulfonate, N,N′-dimethylhydrazine, thioformacetal, formacetal, thioketal, ketal, amine, hydroxylamine, hydroxylamine, or a hydrazinyl group.

In certain embodiments, the linker group comprises an amine. In certain embodiments the linker group comprises polyethylene glycol. In certain embodiments the linker group comprises an ether. In certain embodiments, the linker group comprises a structure as shown below:

wherein m and n are 0 or integers between 1 and 20, and wherein Z_(a) and Z_(b) are each independently a conjugate group, a carbohydrate, a modified carbohydrate, a carbohydrate derivative, H, a peptide, ether, polyethylene glycol, alkyl, C₁-C₂₀ alkyl, substituted C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, substituted C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, substituted C₂-C₂₀ alkynyl, C₁-C₂₀ alkoxy, substituted C₁-C₂₀ alkoxy, amino, amido, pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and 6-aminohexanoic acid.

In certain embodiments, the linker group comprises a structure as shown below:

wherein n is 0 or an integer between 1 and 20, and wherein Z_(a) and Z_(b) are each independently a conjugate group, a carbohydrate, a modified carbohydrate, a carbohydrate derivative, H, a peptide, ether, polyethylene glycol, alkyl, C₁-C₂₀ alkyl, substituted C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, substituted C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, substituted C₂-C₂₀ alkynyl, C₁-C₂₀ alkoxy, substituted C₁-C₂₀ alkoxy, amino, amido, pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and 6-aminohexanoic acid.

In certain embodiments the linker group attaches the conjugate group to the 2′-position of any nucleoside in the oligonucleotide. In certain embodiments the linker group attaches the conjugate group to the phosphate backbone of any nucleoside in the oligonucleotide. In certain embodiments the linker group attaches the conjugate group to the nucleobase of any nucleoside in the oligonucleotide.

In certain embodiments, conjugate groups may be attached to either or both ends of an oligonucleotide (terminal conjugate groups) and/or at any internal position.

In certain embodiments, conjugate groups are at the 3′-end of an oligonucleotide. In certain embodiments, conjugate groups are near the 3′-end. In certain embodiments, conjugate groups are attached at the 3′ end of an oligonucleotide, but before one or more terminal group nucleosides. In certain embodiments, conjugate groups are placed within a terminal group. In certain embodiments, a conjugate group is attached to the 3′-terminal nucleoside. In certain such embodiment, the conjugate group is attached at the 3′-position of the 3′-terminal nucleoside. In certain embodiments, the conjugate group is attached at the 2′-position of the 3′-terminal nucleoside.

In certain embodiments, compounds comprise an oligonucleotide. In certain embodiments, compounds comprises an oligonucleotide and one or more conjugate group and/or terminal groups. Such conjugate group and/or terminal groups may be added to oligonucleotides having any of the chemical motifs discussed above. Thus, for example, a compound comprising an oligonucleotide having region of alternating nucleosides may comprise a terminal group.

In certain embodiments, a conjugate group is attached at the 2′-position of a nucleoside. In certain embodiments, a conjugate group is attached at the 3′-position of the 5′-terminal or 3′-terminal nucleoside. In certain embodiments, the conjugate group is attached at any point along the oligomer's phosphate backbone. In certain embodiments, the conjugate group is attached to the nucleobase.

F. SELECTIVE PLACEMENT OF CERTAIN CONJUGATE GROUPS

In certain embodiments, oligonucleotides are modified by attachment of one or more conjugate groups. In general, conjugate groups modify one or more properties of the attached oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, protein binding, absorption, cellular distribution, cellular uptake, charge, accumulation in specific tissues or organs, and clearance.

In certain embodiments, the selective placement of one or more conjugate groups at various positions along the oligonucleotide serve to enhance one or more properties of the oligonucleotide-conjugate complex. In certain embodiments, the selective placement of one or more conjugate groups at various nucleobases along the oligonucleotide serve to enhance one or more properties of the oligonucleotide-conjugate complex. In general, the selective placement of a conjugate group may enhance the pharmacodynamics, pharmacokinetics, stability, binding, absorption, cellular distribution, cellular uptake, charge, clearance, and other desired properties of the oligonucleotide-conjugate complex.

In certain embodiments, the selective placement of one or more conjugate groups onto an oligonucleotide enhances the nuclease resistance of the oligonucleotide-conjugate complex. In certain embodiments, the selective placement of one or more conjugate groups onto an oligonucleotide enhances the distribution of the oligonucleotide-conjugate complex in one or more organs or tissues. In certain embodiments, the selective placement of one or more conjugate groups onto an oligonucleotide enhances the accumulation of the oligonucleotide-conjugate complex in the liver. In certain embodiments, the selective placement of one or more conjugate groups onto an oligonucleotide enhances the accumulation of the oligonucleotide-conjugate complex in the kidney. In certain embodiments, the selective placement of one or more conjugate groups onto an oligonucleotide enhances the accumulation of the oligonucleotide-conjugate complex in the spleen. In certain embodiments, the selective placement of one or more conjugate groups onto an oligonucleotide enhances the accumulation of the oligonucleotide-conjugate complex in the pancreas. In certain embodiments, the selective placement of one or more conjugate groups onto an oligonucleotide enhances the accumulation of the oligonucleotide-conjugate complex in the adipose tissue.

In certain embodiments, the selective placement of one or more conjugate groups onto an oligonucleotide enhances the accumulation of the oligonucleotide-conjugate complex in a cell. In certain embodiments, the selective placement of one or more conjugate groups onto an oligonucleotide enhances the accumulation of the oligonucleotide-conjugate complex in the nucleus. In certain embodiments, the selective placement of one or more conjugate groups onto an oligonucleotide enhances the accumulation of the oligonucleotide-conjugate complex in the cytoplasm.

In certain embodiments, one or more conjugate groups may be attached at any position on any nucleoside in an oligonucleotide. For example, in certain embodiments, the disclosure provides oligonucleotides consisting of 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, or 14 to 30 linked nucleosides.

In certain embodiments, the oligonucleotide consists of 14 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 nucleoside from the 5′ end or the 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end. In certain embodiments, the oligonucleotide consists of 15 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleoside from the 5′ end or the 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end. In certain embodiments, the oligonucleotide consists of 16 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleoside from the 5′ end or the 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end. In certain embodiments, the oligonucleotide consists of 17 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleoside from the 5′ end or the 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end. In certain embodiments, the oligonucleotide consists of 18 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleoside from the 5′ end or the 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end. In certain embodiments, the oligonucleotide consists of 19 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucleoside from the 5′ end or the 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end.

In certain embodiments, the oligonucleotide consists of 20 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleoside from the 5′ end or the 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end. In certain embodiments, the oligonucleotide consists of 21 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleoside from the 5′ end or the 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end. In certain embodiments, the oligonucleotide consists of 22 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 nucleoside from the 5′ end or the 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end. In certain embodiments, the oligonucleotide consists of 23 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleoside from the 5′ end or the 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end. In certain embodiments, the oligonucleotide consists of 24 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleoside from the 5′ end or the 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end. In certain embodiments, the oligonucleotide consists of 25 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleoside from the 5′ end or the 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end.

In certain embodiments, the oligonucleotide consists of 26 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleoside from the 5′ end or the 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end. In certain embodiments, the oligonucleotide consists of 27 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleoside from the 5′ end or the 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end. In certain embodiments, the oligonucleotide consists of 28 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleoside from the 5′ end or the 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end. In certain embodiments, the oligonucleotide consists of 29 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleoside from the 5′ end or the 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end. In certain embodiments, the oligonucleotide consists of 30 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleoside from the 5′ end or the 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end.

In certain embodiments, a conjugate group is attached to a nucleoside at one or more of position 1, 6 or 8 of the oligonucleotide, counting from the 5′-end. In certain embodiments a conjugate group is attached to a nucleoside at one or more of: position 13, 15, or 20 of the oligonucleotide, counting from the 3′-end.

In certain embodiments, conjugate groups interrupt motifs. For example, in certain embodiments, oligonucleotides of the present disclosure have an alternating motif that spans positions 1-19 and a conjugate group at position 8 (from the 5′-end) as follows:

Po-ABABABAXABABABABABA-

Wherein A represents nucleosides of a first-type;

B represents nucleosides of a second type; and

X represents a nucleoside to which a conjugate group is attached.

In certain embodiments, A and B are 2′-modifications and X is a conjugate group attached at the 2′-position. Thus, the motif of alternating 2′-modifications is interrupted by the conjugate group. Such an oligonucleotide may, nevertheless be described as having an alternating motif.

G. SELECTIVE PLACEMENT OF TWO OR MORE CONJUGATE GROUPS

In certain embodiments, oligonucleotides are modified by attachment of two or more conjugate groups. In general, each of the two or more conjugate groups may modify one or more properties of the attached oligonucleotide, including, but not limited to, the pharmacodynamics, pharmacokinetics, stability, binding, absorption, cellular distribution, cellular uptake, charge and clearance. In certain embodiments, one conjugate group may be selected to enhance the pharmacodynamic, pharmacokinetic, stability, binding, absorption, cellular distribution, cellular uptake, or charge and clearance properties of the attached oligonucleoside, and a second conjugate group may also be selected further to modify the pharmacodynamic, pharmacokinetic, stability, binding, absorption, cellular distribution, cellular uptake, or charge and clearance properties of the attached oligonucleoside. For example, in certain embodiments, a first conjugate group may modify the pharmacokinetic properties of the attached oligonucleotide and a second conjugate group may modify the stability of the attached oligonucleotide, resulting in an oligonucleotide-conjugate complex having both modified pharmacokinetic properties and modified stability.

In certain embodiments, the selective placement of two or more conjugate groups at various positions along the oligonucleotide serve to enhance one or more properties of the oligonucleotide-conjugate complex. In general, the selective placement of two or more conjugate groups may enhance the pharmacodynamics, pharmacokinetics, stability, binding, absorption, cellular distribution, cellular uptake, charge, clearance, and other desired properties of the oligonucleotide-conjugate complex.

In certain embodiments, the selective placement of two or more conjugate groups onto an oligonucleotide enhances the nuclease resistance of the oligonucleotide-conjugate complex. In certain embodiments, the selective placement of two or more conjugate groups onto an oligonucleotide enhances the distribution of the oligonucleotide-conjugate complex in one or more organs or tissues. In certain embodiments, the selective placement of two or more conjugate groups onto an oligonucleotide enhances the accumulation of the oligonucleotide-conjugate complex in the liver. In certain embodiments, the selective placement of two or more conjugate groups onto an oligonucleotide enhances the accumulation of the oligonucleotide-conjugate complex in the kidney. In certain embodiments, the selective placement of two or more conjugate groups onto an oligonucleotide enhances the accumulation of the oligonucleotide-conjugate complex in the spleen. In certain embodiments, the selective placement of two or more conjugate groups onto an oligonucleotide enhances the accumulation of the oligonucleotide-conjugate complex in the pancreas.

In certain embodiments, the selective placement of two or more conjugate groups onto an oligonucleotide enhances the accumulation of the oligonucleotide-conjugate complex in a cell. In certain embodiments, the selective placement of two or more conjugate groups onto an oligonucleotide enhances the accumulation of the oligonucleotide-conjugate complex in the nucleus. In certain embodiments, the selective placement of two or more conjugate groups onto an oligonucleotide enhances the accumulation of the oligonucleotide-conjugate complex in the cytoplasm.

In certain embodiments, two or more conjugate groups may be attached at any position on any nucleoside in an oligonucleotide. For example, in certain embodiments, the disclosure provides oligonucleotides consisting of 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, or 14 to 30 linked nucleosides.

In certain embodiments, the oligonucleotide consists of 14 nucleosides and a conjugate group is attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 nucleoside from the 5′ end or the 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end and a second conjugate group is attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 nucleoside from the 5′ end or the 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end. In certain embodiments, the oligonucleotide consists of 15 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleoside from the 5′ end or the 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end and a second conjugate group is attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleoside from the 5′ end or the 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end. In certain embodiments, the oligonucleotide consists of 16 nucleosides and a conjugate group is attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleoside from the 5′ end or the 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end and a second conjugate group is attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleoside from the 5′ end or the 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end. In certain embodiments, the oligonucleotide consists of 17 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleoside from the 5′ end or the 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end and a second conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleoside from the 5′ end or the 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end. In certain embodiments, the oligonucleotide consists of 18 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleoside from the 5′ end or the 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end and a second conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleoside from the 5′ end or the 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end. In certain embodiments, the oligonucleotide consists of 19 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucleoside from the 5′ end or the 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end and a second conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucleoside from the 5′ end or the 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end.

In certain embodiments, the oligonucleotide consists of 20 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleoside from the 5′ end or the 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end and a second conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleoside from the 5′ end or the 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end. In certain embodiments, the oligonucleotide consists of 21 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleoside from the 5′ end or the 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end and a second conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleoside from the 5′ end or the 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end. In certain embodiments, the oligonucleotide consists of 22 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 nucleoside from the 5′ end or the 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end and a second conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 nucleoside from the 5′ end or the 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end. In certain embodiments, the oligonucleotide consists of 23 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleoside from the 5′ end or the 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end and a second conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleoside from the 5′ end or the 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end. In certain embodiments, the oligonucleotide consists of 24 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleoside from the 5′ end or the 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end and a second conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleoside from the 5′ end or the 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end. In certain embodiments, the oligonucleotide consists of 25 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleoside from the 5′ end or the 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end and a second conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleoside from the 5′ end or the 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end.

In certain embodiments, the oligonucleotide consists of 26 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleoside from the 5′ end or the 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end and a second conjugate group is attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleoside from the 5′ end or the 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end. In certain embodiments, the oligonucleotide consists of 27 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleoside from the 5′ end or the 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end and a second conjugate group is attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleoside from the 5′ end or the 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end. In certain embodiments, the oligonucleotide consists of 28 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleoside from the 5′ end or the 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end and a second conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleoside from the 5′ end or the 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end. In certain embodiments, the oligonucleotide consists of 29 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleoside from the 5′ end or the 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end and a second conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleoside from the 5′ end or the 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end. In certain embodiments, the oligonucleotide consists of 30 nucleosides and a conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleoside from the 5′ end or the 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end and a second conjugate group attached to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleoside from the 5′ end or the 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside from the 3′ end.

In certain embodiments, a conjugate group is attached to a nucleoside at position 8 from the 5′ end and a second conjugate group is attached to the nucleoside at position 6 from the 5′ end. In certain embodiments, a conjugate group is attached to a nucleoside at position 1 from the 5′ end and a second conjugate group is attached to the nucleoside at position 6 from the 5′ end. In certain embodiments, a conjugate group is attached to a nucleoside at position 1 from the 5′ end and a second conjugate group is attached to the nucleoside at position 8 from the 5′ end. In certain embodiments, a conjugate group is attached to a nucleoside at position 21 from the 5′ end and a second conjugate group is attached to the nucleoside at position 6 from the 5′ end. In certain embodiments, a conjugate group is attached to a nucleoside at position 21 from the 5′ end and a second conjugate group is attached to the nucleoside at position 8 from the 5′ end. In certain embodiments, a conjugate group is attached to a nucleoside at position 1 from the 5′ end and a second conjugate group is attached to the nucleoside at position 21 from the 5′ end.

H. ANTISENSE COMPOUNDS

In certain embodiments, compounds of the present disclosure are antisense compounds. Such antisense compounds are capable of hybridizing to a target nucleic acid, resulting in at least one antisense activity. In certain embodiments, antisense compounds specifically hybridize to one or more target nucleic acid. In certain embodiments, a specifically hybridizing antisense compound has a nucleobase sequence comprising a region having sufficient complementarity to a target nucleic acid to allow hybridization and result in antisense activity and insufficient complementarity to any non-target so as to avoid or reduce non-specific hybridization to non-target nucleic acid sequences under conditions in which specific hybridization is desired (e.g., under physiological conditions for in vivo or therapeutic uses, and under conditions in which assays are performed in the case of in vitro assays). In certain embodiments, oligonucleotides are selective between a target and non-target, even though both target and non-target comprise the target sequence. In such embodiments, selectivity may result from relative accessability of the target region of one nucleic acid molecule compared to the other.

In certain embodiments, the present disclosure provides antisense compounds comprising oligonucleotides that are fully complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain embodiments, oligonucleotides are 99% complementary to the target nucleic acid. In certain embodiments, oligonucleotides are 95% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 90% complementary to the target nucleic acid.

In certain embodiments, such oligonucleotides are 85% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 80% complementary to the target nucleic acid. In certain embodiments, an antisense compound comprises a region that is fully complementary to a target nucleic acid and is at least 80% complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain such embodiments, the region of full complementarity is from 6 to 14 nucleobases in length.

In certain embodiments, oligonucleotides comprise a hybridizing region and a terminal region. In certain such embodiments, the hybridizing region consists of 12-30 linked nucleosides and is fully complementary to the target nucleic acid. In certain embodiments, the hybridizing region includes one mismatch relative to the target nucleic acid. In certain embodiments, the hybridizing region includes two mismatches relative to the target nucleic acid. In certain embodiments, the hybridizing region includes three mismatches relative to the target nucleic acid. In certain embodiments, the hybridizing region includes four mismatches relative to the target nucleic acid. In certain embodiments, the terminal region consists of 1-4 terminal nucleosides. In certain embodiments, the terminal nucleosides are at the 3′ end. In certain embodiments, one or more of the terminal nucleosides are not complementary to the target nucleic acid.

Antisense mechanisms include any mechanism involving the hybridization of an oligonucleotide with target nucleic acid, wherein the hybridization results in a biological effect. In certain embodiments, such hybridization results in either target nucleic acid degradation or occupancy with concomitant inhibition or stimulation of the cellular machinery involving, for example, translation, transcription, or splicing of the target nucleic acid.

One type of antisense mechanism involving degradation of target RNA is RNase H mediated antisense. RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are “DNA-like” elicit RNase H activity in mammalian cells. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of DNA-like oligonucleotide-mediated inhibition of gene expression.

Antisense mechanisms also include, without limitation RNAi mechanisms, which utilize the RISC pathway. Such RNAi mechanisms include, without limitation siRNA, ssRNA and microRNA mechanisms.

In certain embodiments, antisense compounds of the present disclosure are RNAi compounds. In certain embodiments, antisense compounds of the present disclosure are ssRNA compounds. In certain embodiments, antisense compounds of the present disclosure are paired with a second oligonucleotide to form an siRNA. In certain such embodiments, the second oligonucleotide is also a compound of the present disclosure. In certain embodiments, the second oligonucleotide is any modified or oligonucleotide. In certain embodiments, the oligonucleotide of the present disclosure is the antisense strand in an siRNA compound. In certain embodiments, the oligonucleotide of the present disclosure is the sense strand in an siRNA compound.

i. Single-Stranded RNAi Compounds

In certain embodiments, oligonucleotides of the present disclosure are particularly suited for use as single-stranded antisense compounds. In certain such embodiments, such oligonucleotides are single-stranded RNAi compounds. In certain embodiments, such oligonucleotides are ssRNA compounds or microRNA mimics. Certain 5′-terminal nucleosides described herein are suited for use in such single-stranded oligonucleotides. In certain embodiments, such 5′-terminal nucleosides stabilize the 5′-phosphorous moiety. In certain embodiments, 5′-terminal nucleosides of the present disclosure are resistant to nucleases. In certain embodiments, the motifs of the present disclosure are particularly suited for use in single-stranded oligonucleotides. For further description of single-stranded RNAi compounds, see, e.g., WO 2010/048585, WO 2010/048549, and PCT/US2011/033968.

Use of single-stranded RNAi compounds has been limited. In certain instances, single stranded RNAi compounds are quickly degraded and/or do not load efficiently into RISC. Design of single-stranded RNAi compounds for use in cells and/or for use in vivo presents several challenges. For example, the compound must be chemically stable, resistant to nuclease degradation, capable of entering cells, capable of loading into RISC (e.g., binding Ago1 or Ago2), capable of hybridizing with a target nucleic acid, and not toxic to cells or animals. In certain instances, a modification or motif that improves one such feature may worsen another feature, rendering a compound having such modification or motif unsuitable for use as an RNAi compound. For example, certain modifications, particularly if placed at or near the 5′-end of an oligonucleotide, may make the compound more stable and more resistant to nuclease degradation, but may also inhibit or prevent loading into RISC by blocking the interaction with RISC components, such as Ago1 or Ago2. Despite its improved stability properties, such a compound would be unsuitable for use in RNAi.

In certain instances, a single-stranded oligonucleotide comprising a 5′-phosphorous moiety is desired. For example, in certain embodiments, such 5′-phosphorous moiety is necessary or useful for RNAi compounds, particularly, single-stranded RNAi compounds. In such instances, it is further desirable to stabilize the phosphorous moiety against degradation or de-phosphorylation, which may inactivate the compound. Further, it is desirable to stabilize the entire 5′-nucleoside from degradation, which could also inactivate the compound. Thus, in certain embodiments, oligonucleotides in which both the 5′-phosphorous moiety and the 5′-nucleoside have been stabilized are desired. In certain embodiments, provided are modified nucleosides that may be placed at the 5′-end of an oligonucleotide, resulting in a stabilized phosphorous and stabilized nucleoside. In certain such embodiments, the phosphorous moiety is resistant to removal in biological systems, relative to unmodified nucleosides and/or the 5′-nucleoside is resistant to cleavage by nucleases. In certain embodiments, such nucleosides are modified at one, at two or at all three of: the 2′-position, the 5′-position, and at the phosphorous moiety. Such modified nucleosides may be incorporated at the 5′-end of an oligonucleotide.

Although certain oligonucleotides described herein have particular use as single-stranded compounds, such compounds may also be paired with a second strand to create a double-stranded compound. In such embodiments, the second strand of the double-stranded duplex may or may not also be an oligonucleotide as described herein.

In some embodiments, oligonucleotides described herein comprise a double stranded composition comprising a first oligomeric compound and a second oligomeric compound, wherein the first oligomeric compound is partially complementary to the second oligomeric compound and the second oligomeric compound is complementary to a nucleic acid target, and wherein at least one of the first and second oligomeric compounds is an oligomeric compound according to any one of the oligonucleotides described herein.

In certain embodiments, such oligonucleotides may be paired with a second strand to form a double-stranded compound. In such embodiments, the second strand of such double-stranded compounds may comprise a motif of the present invention, may comprise another motif of modifications, may be of any suitable length, or may be unmodified. In certain embodiments, such oligonucleotides may be paired with a second strand to form a double-stranded RNAi compound. In such embodiments, the second strand of such double-stranded RNAi compounds may comprise a motif of the present invention, may comprise another motif of modifications, may be of any suitable length, or may be unmodified.

In certain embodiments, oligonucleotides as described herein interact with an argonaute protein (Ago). In certain embodiments, such oligonucleotides first enter the RISC pathway by interacting with another member of the pathway (e.g., dicer). In certain embodiments, oligonucleotides first enter the RISC pathway by interacting with Ago. In certain embodiments, such interaction ultimately results in antisense activity. In certain embodiments, provided are methods of activating Ago comprising contacting Ago with an oligonucleotide. In certain embodiments, such oligonucleotides comprise a modified 5′-phosphate group. In certain embodiments, provided are methods of modulating the expression or amount of a target nucleic acid in a cell comprising contacting the cell with an oligonucleotide capable of activating Ago, ultimately resulting in cleavage of the target nucleic acid. In certain embodiments, the cell is in an animal. In certain embodiments, the cell is in vitro. In certain embodiments, the methods are performed in the presence of manganese. In certain embodiments, the manganese is endogenous. In certain embodiment the methods are performed in the absence of magnesium. In certain embodiments, the Ago is endogenous to the cell. In certain such embodiments, the cell is in an animal. In certain embodiments, the Ago is human Ago. In certain embodiments, the Ago is Ago2. In certain embodiments, the Ago is human Ago2.

In certain embodiments, provided are oligonucleotides having motifs (nucleoside motifs and/or linkage motifs) that result in improved properties. Certain such motifs result in single-stranded oligonucleotides with improved stability and/or cellular uptake properties while retaining antisense activity. For example, oligonucleotides having an alternating nucleoside motif and seven phosphorothioate linkages at to 3′-terminal end have improved stability and activity. Similar compounds that comprise phosphorothioate linkages at each linkage have further improved stability, but are not active as RNAi compounds, presumably because the additional phosphorothioate linkages interfere with the interaction of the oligonucleotide with the RISC pathway components (e.g., with Ago). In certain embodiments, the oligonucleotides having motifs herein result in single-stranded RNAi compounds having desirable properties. In certain embodiments, such oligonucleotides may be paired with a second strand to form a double-stranded RNAi compound. In such embodiments, the second strand of such double-stranded RNAi compounds may comprise a motif as described herein, may comprise another motif of modifications or may be unmodified.

It has been shown that in certain circumstances for single-stranded RNA comprising a 5′-phosphate group has RNAi activity but has much less RNAi activity if it lacks such 5′-phosphate group. The present inventors have recognized that in certain circumstances unmodified 5′-phosphate groups may be unstable (either chemically or enzymatically). Accordingly, in certain circumstances, it is desirable to modify the oligonucleotide to stabilize the 5′-phosphate. In certain embodiments, this is achieved by modifying the phosphate group. In certain embodiments, this is achieved by modifying the sugar of the 5′-terminal nucleoside. In certain embodiments, this is achieved by modifying the phosphate group and the sugar. In certain embodiments, the sugar is modified at the 5′-position, the 2′-position, or both the 5′-position and the 2′-position. As with motifs, above, in embodiments in which RNAi activity is desired, a phosphate stabilizing modification must not interfere with the ability of the oligonucleotide to interact with RISC pathway components (e.g., with Ago).

In certain embodiments, provided are oligonucleotides comprising a phosphate-stabilizing modification and a motif described herein. In certain embodiments, such oligonucleotides are useful as single-stranded RNAi compounds having desirable properties. In certain embodiments, such oligonucleotides may be paired with a second strand to form a double-stranded RNAi compound. In such embodiments, the second strand may comprise a motif as described herein, may comprise another motif of modifications or may be unmodified RNA.

In certain embodiments, provided are compounds and methods for antisense activity in a cell. In certain embodiments, the cell is in an animal. In certain embodiments, the animal is a human. In certain embodiments, provided are methods of administering a compound as described herein to an animal to modulate the amount or activity or function of one or more target nucleic acid.

In certain embodiments oligonucleotides comprise one or more motifs as described herein, but do not comprise a phosphate stabilizing modification. In certain embodiments, such oligonucleotides are useful for in vitro applications.

I. CERTAIN PHARMACEUTICAL COMPOSITIONS

In certain embodiments, the present disclosure provides pharmaceutical compositions comprising one or more antisense compound. In certain embodiments, such pharmaceutical composition comprises a suitable pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutical composition comprises a sterile saline solution and one or more antisense compound. In certain embodiments, such pharmaceutical composition consists of a sterile saline solution and one or more antisense compound. In certain embodiments, the sterile saline is pharmaceutical grade saline. In certain embodiments, a pharmaceutical composition comprises one or more antisense compound and sterile water. In certain embodiments, a pharmaceutical composition consists of one or more antisense compound and sterile water. In certain embodiments, the sterile saline is pharmaceutical grade water. In certain embodiments, a pharmaceutical composition comprises one or more antisense compound and phosphate-buffered saline (PBS). In certain embodiments, a pharmaceutical composition consists of one or more antisense compound and sterile phosphate-buffered saline (PBS). In certain embodiments, the sterile saline is pharmaceutical grade PBS.

In certain embodiments, antisense compounds may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

Pharmaceutical compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters. In certain embodiments, pharmaceutical compositions comprising antisense compounds comprise one or more oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at one or both ends of an oligonucleotide which are cleaved by endogenous nucleases within the body, to form the active antisense oligonucleotide.

Lipid moieties have been used in nucleic acid therapies in a variety of methods. In certain such methods, the nucleic acid is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In certain methods, DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.

In certain embodiments, pharmaceutical compositions provided herein comprise one or more modified oligonucleotides and one or more excipients. In certain such embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.

In certain embodiments, a pharmaceutical composition provided herein comprises a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.

In certain embodiments, a pharmaceutical composition provided herein comprises one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present disclosure to specific tissues or cell types. For example, in certain embodiments, pharmaceutical compositions include liposomes coated with a tissue-specific antibody.

In certain embodiments, a pharmaceutical composition provided herein comprises a co-solvent system. Certain of such co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/v polyethylene glycol 300. The proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.

In certain embodiments, a pharmaceutical composition provided herein is prepared for oral administration. In certain embodiments, pharmaceutical compositions are prepared for buccal administration.

In certain embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, etc.). In certain of such embodiments, a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, such suspensions may also contain suitable stabilizers or agents that increase the solubility of the pharmaceutical agents to allow for the preparation of highly concentrated solutions.

In certain embodiments, a pharmaceutical composition is prepared for transmucosal administration. In certain of such embodiments penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

In certain embodiments, a pharmaceutical composition provided herein comprises an oligonucleotide in a therapeutically effective amount. In certain embodiments, the therapeutically effective amount is sufficient to prevent, alleviate or ameliorate symptoms of a disease or to prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.

In certain embodiments, one or more modified oligonucleotide provided herein is formulated as a prodrug. In certain embodiments, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically more active form of an oligonucleotide. In certain embodiments, prodrugs are useful because they are easier to administer than the corresponding active form. For example, in certain instances, a prodrug may be more bioavailable (e.g., through oral administration) than is the corresponding active form. In certain instances, a prodrug may have improved solubility compared to the corresponding active form. In certain embodiments, prodrugs are less water soluble than the corresponding active form. In certain instances, such prodrugs possess superior transmittal across cell membranes, where water solubility is detrimental to mobility. In certain embodiments, a prodrug is an ester. In certain such embodiments, the ester is metabolically hydrolyzed to carboxylic acid upon administration. In certain instances the carboxylic acid containing compound is the corresponding active form. In certain embodiments, a prodrug comprises a short peptide (polyaminoacid) bound to an acid group. In certain of such embodiments, the peptide is cleaved upon administration to form the corresponding active form.

In certain embodiments, the present disclosure provides compositions and methods for reducing the amount or activity of a target nucleic acid in a cell. In certain embodiments, the cell is in an animal. In certain embodiments, the animal is a mammal. In certain embodiments, the animal is a rodent. In certain embodiments, the animal is a primate. In certain embodiments, the animal is a non-human primate. In certain embodiments, the animal is a human.

In certain embodiments, the present disclosure provides methods of administering a pharmaceutical composition comprising an oligonucleotide of the present disclosure to an animal. Suitable administration routes include, but are not limited to, oral, rectal, transmucosal, intestinal, enteral, topical, suppository, through inhalation, intrathecal, intracerebroventricular, intraperitoneal, intranasal, intraocular, intratumoral, and parenteral (e.g., intravenous, intramuscular, intramedullary, and subcutaneous). In certain embodiments, pharmaceutical intrathecals are administered to achieve local rather than systemic exposures. For example, pharmaceutical compositions may be injected directly in the area of desired effect (e.g., into the liver).

J. CERTAIN USES AND ROUTES OF ADMINISTRATION

In certain embodiments, provided herein are methods of contacting a cell with an oligonucleotide described herein. In certain embodiments, the cell is in vitro. In certain embodiments, the cell is in an animal (e.g., rodent, primate, monkey or human). In certain embodiments, antisense activity is detected.

In certain embodiments, the disease is any of atrophin 1 (DRPLA), Huntington's Disease, Huntington disease-like 2 (HDL2), spinal and bulbar muscular atrophy, Kennedy disease, spinocerebellar ataxia 1, spinocerebellar ataxia 12, spinocerebellar ataxia 17, Huntington disease-like 4 (HDL4), spinocerebellar ataxia 2, spinocerebellar ataxia 3, Machado-Joseph disease, spinocerebellar ataxia 6, spinocerebellar ataxia 7 (OPCA3), ataxin 8 opposite strand (ATXN8OS), myotonic dystrophy (DM1), DM2, and spinocerebellar ataxia 8.

In certain embodiments, compounds as described herein are administered to an animal (e.g., a human) to provide a therapeutic effect. In certain embodiments, the disease is selected from among: ataxin 8, atrophin 1, fragile X syndrome, Friedrich's ataxia, Huntington's disease, Huntington's disease-like 2, myotonic dystrophy, spinal and bulbar muscular atrophy, and spinocerebellar ataxia. In certain embodiments, the disease is Huntington's disease. In certain embodiments, the disease is myotonic dystrophy. In certain embodiments, the myotonic dystrophy is myotonic dystrophy type 1. In certain embodiments, the myotonic dystrophy is myotonic dystrophy type 2. In certain embodiments, the disease is spinocerebellar ataxia. In certain embodiments, the spinocerebellar ataxia is spinocerebellar ataxia 10.

In certain embodiments, pharmaceutical compositions as described herein are administered to a subject. In certain embodiments, such pharmaceutical compositions are administered by injection. In certain embodiments, such pharmaceutical compositions are administered by infusion.

In certain embodiments, pharmaceutical compositions are administered by injection or infusion into the CSF. In certain such embodiments, pharmaceutical compositions are administered by direct injection or infusion into the spine. In certain embodiments, pharmaceutical compositions are administered by injection or infusion into the brain. In certain embodiments, pharmaceutical compositions are administered by intrathecal injection or infusion rather than into the spinal cord tissue itself. Without being limited as to theory, in certain embodiments, the antisense compound released into the surrounding CSF and may penetrate into the spinal cord parenchyma. An additional advantage of intrathecal delivery is that the intrathecal route mimics lumbar puncture administration (i.e., spinal tap) already in routine use in humans.

In certain embodiments, pharmaceutical compositions are administered by intracerebroventricular (ICV) injection or infusion. Intracerebroventricular or intraventricular delivery of a pharmaceutical composition comprising one or more oligonucleotide may be performed in any one or more of the brain's ventricles, which are filled with cerebrospinal fluid (CSF). CSF is a clear fluid that fills the ventricles, is present in the subarachnoid space, and surrounds the brain and spinal cord. CSF is produced by the choroid plexuses and via the weeping or transmission of tissue fluid by the brain into the ventricles. The choroid plexus is a structure lining the floor of the lateral ventricle and the roof of the third and fourth ventricles. Certain studies have indicated that these structures are capable of producing 400-600 ccs of fluid per day consistent with an amount to fill the central nervous system spaces four times in a day. In adult humans, the volume of this fluid has been calculated to be from 125 to 150 ml (4-5 oz). The CSF is in continuous formation, circulation and absorption. Certain studies have indicated that approximately 430 to 450 ml (nearly 2 cups) of CSF may be produced every day. Certain calculations estimate that production equals approximately 0.35 ml per minute in adults and 0.15 per minute in infant humans. The choroid plexuses of the lateral ventricles produce the majority of CSF. It flows through the foramina of Monro into the third ventricle where it is added to by production from the third ventricle and continues down through the aqueduct of Sylvius to the fourth ventricle. The fourth ventricle adds more CSF; the fluid then travels into the subarachnoid space through the foramina of Magendie and Luschka. It then circulates throughout the base of the brain, down around the spinal cord and upward over the cerebral hemispheres. The CSF empties into the blood via the arachnoid villi and intracranial vascular sinuses.

In certain embodiments, such pharmaceutical compositions are administered systemically. In certain embodiments, pharmaceutical compositions are administered subcutaneously. In certain embodiments, pharmaceutical compositions are administered intravenously. In certain embodiments, pharmaceutical compositions are administered by intramuscular injection.

In certain embodiments, pharmaceutical compositions are administered both directly to the CSF (e.g., IT and/or ICV injection and/or infusion) and systemically. In certain such embodiments, compounds as described herein have one or more desirable properties making them suitable for such administration. Drug design typically requires a balance of several variables, including, but not limited to: potency, toxicity, stability, tissue distribution, convenience, and cost of a candidate compound. Such balancing is influenced by a number of factors, including the severity and typical duration of the disease treated. For example, greater drug-related toxicity is tolerated for use in treating acute lethal diseases than chronic sub-lethal diseases. In certain embodiments, compounds as described herein will have one or more improved properties compared to similar compounds that lack certain features as described herein. For example, compared to other compounds, the compounds as described herein, may, in certain embodiments, have improved potency or may have similar potency but reduced toxicity and consequently improved therapeutic index. In certain embodiments, compounds as described herein may have improved pharmacokinetics or distribution to a particular desired target tissue.

In certain embodiments, oligonucleotides as described herein are used in cells in vitro. In certain such embodiments, such uses are to identify and/or study repeat-containing nucleic acids and mechanisms surrounding them and associated diseases.

Nonlimiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references, GenBank accession numbers, and the like recited in the present application is incorporated herein by reference in its entirety.

Although the sequence listing accompanying this filing identifies each sequence as either “RNA” or “DNA” as required, in reality, those sequences may be modified with any combination of chemical modifications. One of skill in the art will readily appreciate that such designation as “RNA” or “DNA” to describe modified oligonucleotides is, in certain instances, arbitrary. For example, an oligonucleotide comprising a nucleoside comprising a 2′-OH sugar moiety and a thymine base could be described as a DNA having a modified sugar (2′-OH for the natural 2′-H of DNA) or as an RNA having a modified base (thymine (methylated uracil) for natural uracil of RNA).

Accordingly, nucleic acid sequences provided herein, including, but not limited to those in the sequence listing, are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases. By way of further example and without limitation, an oligonucleotide having the nucleobase sequence “ATCGATCG” encompasses any oligonucleotides having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence “AUCGAUCG” and those having some DNA bases and some RNA bases such as “AUCGATCG” and oligonucleotides having other modified bases, such as “AT^(me)CGAUCG,” wherein ^(me)C indicates a cytosine base comprising a methyl group at the 5-position.

EXAMPLES

The following examples illustrate certain embodiments of the present disclosure and are not limiting. Moreover, where specific embodiments are provided, the inventors have contemplated generic application of those specific embodiments. For example, disclosure of an oligonucleotide having a particular motif provides reasonable support for additional oligonucleotides having the same or similar motif. And, for example, where a particular high-affinity modification appears at a particular position, other high-affinity modifications at the same position are considered suitable, unless otherwise indicated.

Examples

The following examples illustrate certain embodiments of the present disclosure and are not limiting. Moreover, where specific embodiments are provided, the inventors have contemplated generic application of those specific embodiments. For example, disclosure of an oligonucleotide having a particular motif provides reasonable support for additional oligonucleotides having the same or similar motif. And, for example, where a particular high-affinity modification appears at a particular position, other high-affinity modifications at the same position are considered suitable, unless otherwise indicated.

Example 1 Synthesis of Oligomeric Compounds

The oligomeric compounds used in accordance with this disclosure may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as alkylated derivatives and those having phosphorothioate linkages.

Oligomeric compounds: Unsubstituted and substituted phosphodiester (P═O) oligomeric compounds, including without limitation, oligonucleotides can be synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry with oxidation by iodine.

In certain embodiments, phosphorothioate internucleoside linkages (P═S) are synthesized similar to phosphodiester internucleoside linkages with the following exceptions: thiation is effected by utilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the oxidation of the phosphite linkages. The thiation reaction step time is increased to 180 sec and preceded by the normal capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (12-16 hr), the oligomeric compounds are recovered by precipitating with greater than 3 volumes of ethanol from a 1 M NH₄OAc solution. Phosphinate internucleoside linkages can be prepared as described in U.S. Pat. No. 5,508,270.

Alkyl phosphonate internucleoside linkages can be prepared as described in U.S. Pat. No. 4,469,863.

3′-Deoxy-3′-methylene phosphonate internucleoside linkages can be prepared as described in U.S. Pat. No. 5,610,289 or 5,625,050.

Phosphoramidite internucleoside linkages can be prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878.

Alkylphosphonothioate internucleoside linkages can be prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively).

3′-Deoxy-3′-amino phosphoramidate internucleoside linkages can be prepared as described in U.S. Pat. No. 5,476,925.

Phosphotriester internucleoside linkages can be prepared as described in U.S. Pat. No. 5,023,243.

Borano phosphate internucleoside linkages can be prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198.

Oligomeric compounds having one or more non-phosphorus containing internucleoside linkages including without limitation methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone oligomeric compounds having, for instance, alternating MMI and P═O or P═S linkages can be prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289.

Formacetal and thioformacetal internucleoside linkages can be prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564.

Ethylene oxide internucleoside linkages can be prepared as described in U.S. Pat. No. 5,223,618.

Example 2 Isolation and Purification of Oligomeric Compounds

After cleavage from the controlled pore glass solid support or other support medium and deblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours, the oligomeric compounds, including without limitation oligonucleotides and oligonucleosides, are recovered by precipitation out of 1 M NH₄OAc with >3 volumes of ethanol. Synthesized oligomeric compounds are analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis. The relative amounts of phosphorothioate and phosphodiester linkages obtained in the synthesis is determined by the ratio of correct molecular weight relative to the −16 amu product (+/−32 +/−48). For some studies oligomeric compounds are purified by HPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified material are generally similar to those obtained with non-HPLC purified material.

Example 3 Synthesis of Oligomeric Compounds Using the 96 Well Plate Format

Oligomeric compounds, including without limitation oligonucleotides, can be synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 96-well format. Phosphodiester internucleoside linkages are afforded by oxidation with aqueous iodine. Phosphorothioate internucleoside linkages are generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyl-diiso-propyl phosphoramidites can be purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per standard or patented methods and can be functionalized as base protected beta-cyanoethyldiisopropyl phosphoramidites.

Oligomeric compounds can be cleaved from support and deprotected with concentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product is then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.

Example 4 Analysis of Oligomeric Compounds Using the 96-Well Plate Format

The concentration of oligomeric compounds in each well can be assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products can be evaluated by capillary electrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition is confirmed by mass analysis of the oligomeric compounds utilizing electrospray-mass spectroscopy. All assay test plates are diluted from the master plate using single and multi-channel robotic pipettors. Plates are judged to be acceptable if at least 85% of the oligomeric compounds on the plate are at least 85% full length.

Example 5 In Vitro Treatment of Cells with Oligomeric Compounds

The effect of oligomeric compounds on target nucleic acid expression is tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. Cell lines derived from multiple tissues and species can be obtained from American Type Culture Collection (ATCC, Manassas, Va.).

The following cell type is provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen. This can be readily determined by methods routine in the art, for example Northern blot analysis, ribonuclease protection assays or RT-PCR.

b.END cells: The mouse brain endothelial cell line b.END was obtained from Dr. Werner Risau at the Max Plank Institute (Bad Nauheim, Germany). b.END cells are routinely cultured in DMEM, high glucose (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells are routinely passaged by trypsinization and dilution when they reached approximately 90% confluence. Cells are seeded into 96-well plates (Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a density of approximately 3000 cells/well for uses including but not limited to oligomeric compound transfection experiments.

Experiments involving treatment of cells with oligomeric compounds:

When cells reach appropriate confluency, they are treated with oligomeric compounds using a transfection method as described.

Lipofectin™

When cells reached 65-75% confluency, they are treated with one or more oligomeric compounds. The oligomeric compound is mixed with LIPOFECTIN™ Invitrogen Life Technologies, Carlsbad, Calif.) in Opti-MEM™-1 reduced serum medium (Invitrogen Life Technologies, Carlsbad, Calif.) to achieve the desired concentration of the oligomeric compound(s) and a LIPOFECTIN™ concentration of 2.5 or 3 μg/mL per 100 nM oligomeric compound(s). This transfection mixture is incubated at room temperature for approximately 0.5 hours. For cells grown in 96-well plates, wells are washed once with 100 μL OPTI-MEM™-1 and then treated with 130 μL of the transfection mixture. Cells grown in 24-well plates or other standard tissue culture plates are treated similarly, using appropriate volumes of medium and oligomeric compound(s). Cells are treated and data are obtained in duplicate or triplicate. After approximately 4-7 hours of treatment at 37° C., the medium containing the transfection mixture is replaced with fresh culture medium. Cells are harvested 16-24 hours after treatment with oligomeric compound(s).

Other suitable transfection reagents known in the art include, but are not limited to, CYTOFECTIN™, LIPOFECTAMINE™, OLIGOFECTAMINE™, and FUGENE™. Other suitable transfection methods known in the art include, but are not limited to, electroporation.

Example 6 Real-Time Quantitative PCR Analysis of Target mRNA Levels

Quantitation of target mRNA levels is accomplished by real-time quantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM™ Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.

Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be “multiplexed” with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only (“single-plexing”), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art.

RT and PCR reagents are obtained from Invitrogen Life Technologies (Carlsbad, Calif.). RT, real-time PCR is carried out by adding 20 μL PCR cocktail (2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-well plates containing 30 μL total RNA solution (20-200 ng). The RT reaction is carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol are carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

Gene target quantities obtained by RT, real-time PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RIBOGREEN™ (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen™ RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA quantification by RIBOGREEN™ are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).

In this assay, 170 μL of RIBOGREEN™ working reagent (RIBOGREEN™ reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 μL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nm and emission at 530 nm.

Example 7 Analysis of Oligonucleotide Inhibition of Target Expression

Antisense modulation of a target expression can be assayed in a variety of ways known in the art. For example, a target mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR. Real-time quantitative PCR is presently desired. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. One method of RNA analysis of the present disclosure is the use of total cellular RNA as described in other examples herein. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.

Protein levels of a target can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA) or fluorescence-activated cell sorting (FACS). Antibodies directed to a target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art. Methods for preparation of polyclonal antisera are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.

Immunoprecipitation methods are standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998. Western blot (immunoblot) analysis is standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons, Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley & Sons, Inc., 1991.

Example 8 Design of Phenotypic Assays and In Vivo Studies for the Use of Target Inhibitors

Phenotypic Assays

Once target inhibitors have been identified by the methods disclosed herein, the oligomeric compounds are further investigated in one or more phenotypic assays, each having measurable endpoints predictive of efficacy in the treatment of a particular disease state or condition.

Phenotypic assays, kits and reagents for their use are well known to those skilled in the art and are herein used to investigate the role and/or association of a target in health and disease. Representative phenotypic assays, which can be purchased from any one of several commercial vendors, include those for determining cell viability, cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assays including enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.), cell regulation, signal transduction, inflammation, oxidative processes and apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglyceride accumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube formation assays, cytokine and hormone assays and metabolic assays (Chemicon International Inc., Temecula, Calif.; Amersham Biosciences, Piscataway, N.J.).

In one non-limiting example, cells determined to be appropriate for a particular phenotypic assay (i.e., MCF-7 cells selected for breast cancer studies; adipocytes for obesity studies) are treated with a target inhibitors identified from the in vitro studies as well as control compounds at optimal concentrations which are determined by the methods described above. At the end of the treatment period, treated and untreated cells are analyzed by one or more methods specific for the assay to determine phenotypic outcomes and endpoints.

Phenotypic endpoints include changes in cell morphology over time or treatment dose as well as changes in levels of cellular components such as proteins, lipids, nucleic acids, hormones, saccharides or metals. Measurements of cellular status which include pH, stage of the cell cycle, intake or excretion of biological indicators by the cell, are also endpoints of interest.

Measurement of the expression of one or more of the genes of the cell after treatment is also used as an indicator of the efficacy or potency of the a target inhibitors. Hallmark genes, or those genes suspected to be associated with a specific disease state, condition, or phenotype, are measured in both treated and untreated cells.

In Vivo Studies

The individual subjects of the in vivo studies described herein are warm-blooded vertebrate animals, which includes humans.

Example 9 RNA Isolation

Poly(A)+ mRNA Isolation

Poly(A)+ mRNA is isolated according to Miura et al., (Clin. Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation are routine in the art. Briefly, for cells grown on 96-well plates, growth medium is removed from the cells and each well is washed with 200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) is added to each well, the plate is gently agitated and then incubated at room temperature for five minutes. 55 of lysate is transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates are incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate is blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C., is added to each well, the plate is incubated on a 90° C. hot plate for 5 minutes, and the eluate is then transferred to a fresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.

Total RNA Isolation

Total RNA is isolated using an RNEASY 96™ kit and buffers purchased from Qiagen Inc. (Valencia, Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium is removed from the cells and each well is washed with 200 μL cold PBS. 150 μL Buffer RLT is added to each well and the plate vigorously agitated for 20 seconds. 150 μL of 70% ethanol is then added to each well and the contents mixed by pipetting three times up and down. The samples are then transferred to the RNEASY 96™ well plate attached to a QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum is applied for 1 minute. 500 μL of Buffer RW1 is added to each well of the RNEASY 96™ plate and incubated for 15 minutes and the vacuum is again applied for 1 minute. An additional 500 μL of Buffer RW1 is added to each well of the RNEASY 96™ plate and the vacuum is applied for 2 minutes. 1 mL of Buffer RPE is then added to each well of the RNEASY 96™ plate and the vacuum applied for a period of 90 seconds. The Buffer RPE wash is then repeated and the vacuum is applied for an additional 3 minutes. The plate is then removed from the QIAVAC™ manifold and blotted dry on paper towels. The plate is then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA is then eluted by pipetting 140 μL of RNAse free water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.

The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.

Example 10 Target-Specific Primers and Probes

Probes and primers may be designed to hybridize to a target sequence, using published sequence information.

For example, for human PTEN, the following primer-probe set was designed using published sequence information (GENBANK™ accession number U92436.1, SEQ ID NO: 1).

(SEQ ID NO: 2) Forward primer: AATGGCTAAGTGAAGATGACAATCAT (SEQ ID NO: 3) Reverse primer: TGCACATATCATTACACCAGTTCGT And the PCR probe:

(SEQ ID NO: 4) FAM-TTGCAGCAATTCACTGTAAAGCTGGAAAGG-TAMRA, where FAM is the fluorescent dye and TAMRA is the quencher dye.

Example 11 Western Blot Analysis of Target Protein Levels

Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 μl/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to a target is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

Example 12 General Method for the Preparation of Oligomeric Compounds Containing Modified Nucleosides as Provided Herein at the 5′-Terminus Via Solid Phase Techniques (Preparation of 505739)

Unless otherwise stated, all reagents and solutions used for the synthesis of oligomeric compounds were purchased from commercial sources. Standard phosphoramidites and solid support were used for incorporation of A, U, G, ^(me)C and C residues. A 0.1 M solution of 2′-F and 2′-O-Me phosphoramidites in anhydrous acetonitrile (CH₃CN) along with 2′-O-MOE-5′-deoxy-5′-methylenediethylphosphonate and 2′-O-MOE-deoxy-5′-vinyldimethylphosphonate 3′-phosphoramidites in 30% dichloromethane (CH₂Cl₂) in anhydrous CH₃CN were used for the synthesis. The oligomeric compounds were synthesized on VIMAD UnyLinker™ solid support and the appropriate amounts of solid support were packed in the column for synthesis. Dichloroacetic acid (6%) in toluene was used as detritylating reagent. 4,5-Dicyanoimidazole in the presence of N-methylimidazole or 1H-tetrazole in CH₃CN was used as activator during the coupling step. The synthesis of oligomeric compounds was performed either on an ÄKTAOligopilot synthesizer (GE Healthcare Bioscience) or an ABI394 synthesizer (Applied Biosystems) on a 2-200 μmol scale using the procedures set forth below.

A solid support preloaded with the Unylinker™ was loaded into a synthesis column after closing the column bottom outlet and CH₃CN was added to form a slurry. The swelled support-bound Unylinker™ was treated with a detritylating reagent containing 6% dichloroacetic acid in toluene to provide the free hydroxyl groups. During the coupling step, four to fourteen equivalents of phosphoramidite solutions were delivered with coupling for 10 minutes. All of the other steps followed standard protocols. Phosphorothioate linkages were introduced by sulfurization with a 0.05 M solution of DDTT (3-((dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole-3-thione) in 1:1 pyridine/CH₃CN for a contact time of 3 minutes. Phosphite triester internucleoside linkages were oxidized to phosphate diester internucleoside linkages using a solution of tert-butyl hydroperoxide/CH₃CN/water (10:87:3) over 12 minutes.

After the desired sequence was assembled, the solid support bound oligomeric compound was washed with CH₂Cl₂ and dried under high vacuum. After 4 hrs, the dried solid support was suspended in a solution of iodotrimethylsilane (TMSI) and pyridine in CH₂Cl₂ to remove the 5′-phosphonate protecting group (ethyl ether or methyl ether). The deprotection solution was prepared by dissolving 0.75 mL TMSI and 0.53 mL pyridine in 28.2 mL CH₂Cl₂ (used 0.5 mL/μmol of solid support). After 30 min at room temperature, the reaction was quenched with 1M 2-mercaptoethanol in 1:1 TEA/CH₃CN (used 0.5 mL/μmol of solid support). The supernatant was decanted and the solid-support was washed with additional 2-mercaptoethanol solution. After 45 minutes at room temperature the wash step with additional 2-mercaptoethanol solution was repeated. The supernatant was decanted and the solid-support bound oligomeric compound was suspended in ammonia (28-30 wt %) in 1 M 2-mercaptoethanol (used 0.75 mL/μmol of solid support) and heated at 55° C. for 2 hrs to cleave the oligomeric compound from the solid support.

The cleaved solution was allowed to cool to ambient temperature (20° C.) for 24 hrs. The unbound oligomeric compound was then filtered and the support was rinsed and filtered with water:ethanol (1:1) followed by water. The filtrate was combined and concentrated to dryness. The residue obtained was purified by HPCL on a reverse phase column (Waters X-Bridge C-18 5 μm, 19×250 mm, A=5 mM tributylammonium acetate in 5% aqueous CH₃CN, B═CH₃CN, 0 to 90% B in 80 min, flow 7 mL min⁻¹, λ=260 nm). Fractions containing full-length oligomeric compound were pooled together (assessed by LC/MS analysis >95%) and the tributylammonium counter ion was exchanged to sodium by HPLC on a strong anion exchange column (GE Healthcare Bioscience, Source 30Q, 30 μm, 2.54×8 cm, A=100 mM ammonium acetate in 30% aqueous CH₃CN, B=1.5 M NaBr in A, 0-40% of B in 60 min, flow 14 mL min⁻¹). The residue was desalted by HPLC on a reverse phase column to yield the oligomeric compound in an isolated yield of 15-20% based on solid-support loading. The unbound oligomeric compound was characterized by ion-pair-HPLC-MS analysis with Agilent 1100 MSD system.

The modified oligonucleotides comprising a 5′-(E)-vinylphosphonate group is described in Table 1. A subscript “s” between two nucleosides indicates a phosphorothioate internucleoside linkage (going 5′ to 3′). The absence of a subscript “s” between two nucleosides indicates a phosphodiester internucleoside linkage. A “Pv” at the 5′-end indicates a 5′-(E)-vinylphosphonate group, (PO(OH)₂(CH═CH)—. Nucleosides followed by a subscript “f”, “m” or “e” are sugar modified nucleosides. A subscript “f” indicates a 2′-fluoro modified nucleoside, a subscript “m” indicates a 2′-O-methyl modified nucleoside and a subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside.

TABLE 1 Modified oligomeric compound comprising a 5′-(E)-vinyphosphonate SEQ ISIS NO. Composition (5′ to 3′) 5′-Chemistry ID NO. 505739 Pv-T_(es)U_(fs)G_(m)U_(fs)C_(m)U_(fs)C_(m)U_(fs)G_(m)G_(fs) (E)-(PO(OH)₂(CH═CH)— 5 U_(m)C_(fs)C_(m)U_(fs)U_(ms)A_(fs)C_(ms)U_(fs)U_(ms)A_(es)A_(e)

Example 13 Preparation of Modified Oligomeric Compounds Comprising a Modified 5′-Nucleoside (5′-Deoxy-5′-Methylenediethylphosphonate and 5′-Deoxy-5′-Vinyldimethylphosphonate)

Oligomeric compounds were prepared on either a 2 or 200 μmol scale following procedures illustrated in Example 12. Both ÄKTAOligopilot synthesizer (GE Healthcare Bioscience) and ABI394 synthesizer (Applied Biosystems) were used for particular runs. The unbound oligomeric compounds were cleaved from the solid support and analyzed by ion-pair-HPLC-MS.

The modified oligonucleotides comprising a 5′-(E)-vinylphosphonate group, (PO(OH)₂(CH═CH—), and a 5′-methylenediethylphosphonate group, (PO(OCH₂CH₃)₂(CH₂CH₂—) are described in Table 2. A subscript “s” between two nucleosides indicates a phosphorothioate internucleoside linkage (going 5′ to 3′). The absence of a subscript “s” between two nucleosides indicates a phosphodiester internucleoside linkage. A “Py” at the 5′-end indicates a 5′-methylenediethylphosphonate group, (PO(OCH₂CH₃)₂(CH₂CH₂—). A “Pv” at the 5′-end indicates a 5′-(E)-vinylphosphonate group, (PO(OH)₂(CH═CH—). Nucleosides followed by a subscript “f”,” “m” or “e” are sugar modified nucleosides. A subscript “f” indicates a 2′-fluoro modified nucleoside, a subscript “m” indicates a 2′-O-methyl modified nucleoside and a subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside.

TABLE 2 Modified oligonucleotides comprising a modified 5′-terminus group SEQ ISIS NO. Composition (5′ to 3′) 5′-Chemistry ID NO. 508027 Py-T_(es)U_(fs)G_(m)U_(fs)C_(m)U_(fs)C_(m)U_(fs)G_(m)G_(fs) (PO(OCH₂CH₃)₂(CH₂CH₂-) 5 U_(m)C_(fs)C_(m)U_(fs)U_(ms)A_(fs)C_(ms)U_(fs)U_(ms)A_(es)A_(e) 505739 PV-T_(es)U_(fs)G_(m)U_(fs)C_(m)U_(fs)C_(m)U_(fs)G_(m)G_(fs) (E)-(PO(OH)₂(CH═CH-) 5 U_(m)C_(fs)C_(m)U_(fs)U_(ms)A_(fs)C_(ms)U_(fs)U_(ms)A_(es)A_(e) 508016 Py-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs) (PO(OCH₂CH₃)₂(CH₂CH₂-) 6 U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) 522247 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs)U_(m) (E)-(PO(OH)₂(CH═CH-) 6 G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) 508015 Py-T_(es)G_(fs)A_(m)A_(fs)C_(m)A_(fs)U_(m)U_(fs)G_(m)G_(fs) (PO(OCH₂CH₃)₂(CH₂CH₂-) 7 A_(m)A_(fs)U_(m)A_(fs)G_(ms)U_(fs)U_(ms)U_(fs)C_(ms)A_(es)A_(e) 522246 Pv-T_(es)G_(fs)A_(m)A_(fs)C_(m)A_(fs)U_(m)U_(fs)G_(m)G_(fs) (E)-(PO(OH)₂(CH═CH-) 7 A_(m)A_(fs)U_(m)A_(fs)G_(ms)U_(fs)U_(ms)U_(fs)C_(ms)A_(es)A_(e)

Example 14 General Method for the Preparation of C16- and Cholesterol-Conjugated Oligomeric Compounds 2 and 3

Compound 1 and conjugated oligomeric Compounds 2 and 3 are prepared according to published procedures (see Swayze et al., WO 2006/031461) and procedures illustrated in Example 12.

Example 15 Preparation of C16- and Cholesterol-Conjugated Oligomeric Compounds

The C16 and cholesterol conjugated oligomeric compounds shown in Table 3 were prepared as per the procedures illustrated in Examples 12 and 14.

The conjugated oligomeric compounds were cleaved from the solid support, analyzed by ion-pair-HPLC-MS and are described in Table 3. A subscript “s” between two nucleosides indicates a phosphorothioate internucleoside linkage (going 5′ to 3′). The absence of a subscript “s” between two nucleosides indicates a phosphodiester internucleoside linkage. A “Pv” at the 5′-end indicates a 5′-(E)-vinylphosphonate group (PO(OH)₂(CH═CH—). Nucleosides followed by a subscript f, m or e are sugar modified nucleosides. A subscript “f” indicates a 2′-fluoro modified nucleoside, a subscript “m” indicates a 2′-O-methyl modified nucleoside and a subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside. The C16 alkyl chain and cholesterol conjugated through a pyrrolidine linker group are shown below. Underlined nucleosides indicate the conjugate position.

TABLE 3 Modified oligonucleotides comprising conjugates Mass (Da) ISIS NO. Composition (5′ to 3′) Calculated Observed SEQ ID NO. 526608 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs)U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es) A_(e)-C16 7879.2 7877.6 6 527155 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs)U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es) A_(e)-Cholesterol 8067.5 8066.5 6

Example 16 Preparation of C16- and Cholesterol-Conjugated Oligomeric Compounds

Additional C16- and cholesterol-conjugated oligonucleotides comprising a 5′-(E)-vinylphosphonate group were designed based on the parent oligonucleotide, ISIS 522247 and were tested for their ability to reduce PTEN mRNA levels in hepatocytes. The modified oligonucleotides were created with C16 or cholesterol conjugate group at the 3′-terminus. The potency of the 3′-C16 or 3′-cholesterol-conjugated oligonucleotides were evaluated and compared to the parent oligonucleotide lacking a conjugate group, ISIS 522247 or 5-10-5 MOE gapmer, ISIS 116847.

The conjugated oligonucleotides were prepared as per the procedures illustrated in Example 14 and are described in Table 4. A subscript “s” between two nucleosides indicates a phosphorothioate internucleoside linkage (going 5′ to 3′). The absence of a subscript “s” between two nucleosides indicates a phosphodiester internucleoside linkage. A “Pv” at the 5′-end indicates a 5′-(E)-vinylphosphonate group (PO(OH)₂(CH═CH—). Nucleosides followed by a subscript “1”, “m” or “e” are sugar modified nucleosides. A subscript “f” indicates a 2′-fluoro modified nucleoside, a subscript “m” indicates a 2′-O-methyl modified nucleoside and a subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside. Nucleosides not followed by a subscript is β-D-2′-deoxyribonucleoside. ^(me)C indicates a 5-methyl cytosine nucleoside. The C16 alkyl chain and cholesterol conjugated through a pyrrolidine linker group are shown below. Underlined nucleosides indicate the conjugate position.

The 3′-C16 or 3′-cholesterol-conjugated oligonucleotides were tested in vitro. Hepatocytes were treated with the modified oligonucleotides shown in Table 4 using LIPOFECTAMINE™ 2000 (Lipo) as transfection method as described herein. The IC₅₀'s were calculated using the linear regression equation generated by plotting the normalized mRNA levels to the log of the concentrations used and the results are presented in Table 4.

The parent oligomeric compound, ISIS 522247, from which the newly designed oligonucleotides were derived from is marked with an asterisk (*) in the table. The 5-10-5 MOE gapmer, ISIS 116847 was also included in the study as a benchmark oligonucleotide against which the potency of the 3′-C16 or 3′-cholesterol-conjugated oligonucleotides could be compared.

As illustrated in Table 4, modified oligonucleotide comprising a C16 conjugate at the 3′ terminus exhibited comparable potency while the 3′-cholesterol conjugate showed no improvement in potency comparing to the parent oligonucleotide, ISIS 522247 and 5-10-5 MOE gapmer.

TABLE 4 Comparison of inhibition of PTEN mRNA levels of 3′-C16 or 3′-cholesterol-conjugated oligonucleotides with ISIS 116847 and ISIS 522247 ISIS IC₅₀ SEQ ID NO. Composition (5′ to 3′) Chemistry (nM) NO. 116847 ^(me)C_(es)T_(es)G_(es) ^(me)C_(es)T_(es)A_(s)G_(s) ^(me)C_(s) ^(me)C_(s)T_(s) ^(me)C_(s)T_(s)G_(s)G_(s)A_(s)T_(es)T_(es)T_(es)G_(e)A_(e) 5-10-5 1 8 MOE gapmer 522247 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs)U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) 5′-(E)- 0.9 6 (PO(OH)₂(CH═CH)— 526608 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs)U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es) A_(e)-C16 3′-C16 2 6 529106 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs)U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(m)A_(es) A_(e)-Cholesterol 3′-Cholesterol 10 6

Example 17 General Method for the Preparation of C10-Conjugated Oligomeric Compound 6

The Unylinker™ 4 is commercially available. Conjugated oligomeric Compound 6 is prepared similar to published procedures (see Swayze et al., WO 2006/031461) and procedures illustrated in Examples 12-14.

Example 18 Preparation of C10-Conjugated Oligonucleotides

The conjugated oligonucleotides in Table 5 were prepared as per the procedures illustrated in Example 17 comprising a C10 conjugate attached to either at the 5′, 3′ or at any internal positions of the oligomeric compound.

The modified oligonucleotides comprising a 5′-(E)-vinylphosphonate group and a C10 conjugate are described in Table 5. A subscript “s” between two nucleosides indicates a phosphorothioate internucleoside linkage (going 5′ to 3′). The absence of a subscript “s” between two nucleosides indicates a phosphodiester internucleoside linkage. A “Pv” at the 5′-end indicates a 5′-(E)-vinylphosphonate group, (PO(OH)₂(CH═CH—). Nucleosides followed by a subscript “f”, “m” or “e” are sugar modified nucleosides. A subscript “f” indicates a 2′-fluoro modified nucleoside, a subscript “m” indicates a 2′-O-methyl modified nucleoside and a subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside. Nucleosides with a subscript “C10” are shown below. Underlined nucleosides indicate the conjugate position.

Table 5

Modified oligonucleotides comprising C10 conjugate Conjugate position counted from SEQ ID ISIS NO. Composition (5′ to 3′) 5′ 3′ NO. 530340  Pv-T_(es)U_(fs)G_(m)U_(fs)C_(m)U_(fs)C_(m)U_(fs)G_(m)G_(fs)U_(m)C_(fs)C_(m)U_(fs)U_(ms)A_(fs)C_(ms)U_(fs) T_(C10s) A_(es)A_(e) 19  3  9 or341 530334 Pv-T_(es)U_(fs)G_(m)U_(fs)C_(m)U_(fs)C_(m)U_(fs)G_(m)G_(fs)U_(m)C_(fs)C_(m)U_(fs) T_(C10s) A_(fs)C_(ms)U_(fs)U_(ms)A_(es)A_(e) 15  7 10 530335 Pv-T_(es)U_(fs)G_(m)U_(fs)C_(m)U_(fs)C_(m)U_(fs)G_(m)G_(fs) T_(C10) C_(fs)C_(m)U_(fs)U_(ms)A_(fs)C_(ms)U_(fs)U_(ms)A_(es)A_(e) 11 11 11 530336 Pv-T_(es)U_(fs)G_(m)U_(fs)C_(m) T_(C10s) C_(m)U_(fs)G_(m)G_(fs)U_(m)C_(fs)C_(m)U_(fs)U_(ms)A_(fs)C_(ms)U_(fs)U_(ms)A_(es)A_(e)  6 16 12 530337 Pv-T_(es)U_(fs)G_(m)U_(fs)C_(m)U_(fs)C_(m)U_(fs)G_(m)G_(fs)U_(m)C_(fs)C_(m)U_(fs)U_(ms)A_(fs)C_(ms)U_(fs)U_(ms)A_(es) T_(C10) 21  1 13 549621 Pv-T_(C10s) U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs)U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e)  1 21 14

Example 19 Preparation of C10-Conjugated Oligonucleotides

Additional C10-conjugated oligonucleotides comprising a 5′-(E)-vinylphosphonate group were designed based on the parent oligonucleotide, ISIS 522247 from Table 2 in an effort to evaluate the effects of conjugate groups on cellular uptake and potency. The modified oligonucleotides were designed by introducing a C10 conjugate, wherein the C10 conjugate will be shifted slightly upstream or downstream throughout the antisense oligonucleotides (i.e. “microwalk”).

The C10-conjugated oligonucleotides were prepared as per the procedures illustrated in Example 17 and are described in Table 6. A subscript “s” between two nucleosides indicates a phosphorothioate internucleoside linkage (going 5′ to 3′). The absence of a subscript “s” between two nucleosides indicates a phosphodiester internucleoside linkage. A “Pv” at the 5′-end indicates a 5′-(E)-vinylphosphonate group, (PO(OH)₂(CH═CH—). A “Po” at the 5′-end indicates a 5′-phosphate group, (PO(OH)₂)—. Nucleosides followed by a subscript “f”, “m” or “e” are sugar modified nucleosides. A subscript “f” indicates a 2′-fluoro modified nucleoside, a subscript “m” indicates a 2′-O-methyl modified nucleoside and a subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside. Nucleosides with a subscript “C10” are shown below. Underlined nucleosides indicate the conjugate position.

The C10-conjugated oligonucleotides were tested in vitro. HeLa cells and hepatocytes were treated with modified oligonucleotides shown in Table 6 using transfection methods such as LIPOFECTAMINE′ 2000 (Lipo), electroporation (Electro) or free up-take as described herein. The IC₅₀'s were calculated using the linear regression equation generated by plotting the normalized mRNA levels to the log of the concentrations used and the results are presented in Table 7.

The parent oligomeric compound, ISIS 522247, from which the newly designed oligonucleotides were derived from is marked with an asterisk (*) in the table and was included in the study as a benchmark oligonucleotide against which the cellular uptake and potency of the C10-conjugated oligonucleotides could be compared. Oligonucleotides without data are denoted as “ND”.

As illustrated in Table 7, modified oligonucleotides comprising a C10 conjugate group at positions 1, 6 or 8, as counted from the 5′-terminus, or positions 21, 16, or 14, as counted from the 3′-terminus showed comparable potency and were well tolerated for cellular uptake in a similar manner as the parent oligonucleotide lacking a conjugate group, ISIS 522247. The remaining oligonucleotides (ISIS 534711 and ISIS 534713) showed little to no improvement in cellular uptake.

TABLE 6 Modified oligonucleotides comprising C10 conjugate Conjugate position counted from ISIS NO. Composition (5′ to 3′) 5′ 3′ SEQ ID NO.  522247* PV-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs) N/A N/A  6 U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) 534714 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs) 21  1 15 U_(m)G_(fs)A_(m)U_(fs)C_(mS)A_(fs)G_(ms)G_(fs)U_(ms)A_(es) T_(C10) 534711 PV-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs) 19  3 16 U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs) T_(C10s) A_(es)A_(e) 534713 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs) 11 11 17 T_(C10) G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) 533814 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m) T_(C10s) A_(m)A_(fs)  8 14 18 U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) 533815 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m) T_(C10s) A_(m)U_(fs)A_(m)A_(fs)  6 16 19 U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) 533813 Pv-T_(es)U_(fs)A_(m) T_(C10s) C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs)  4 18 20 U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) 533816 Po-T_(es)U_(fs)A_(m) T_(C10s) C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs)  1 21  6 U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e)

TABLE 7 Comparison of inhibition of PTEN mRNA levels of  C10-conjugated oligonucleotides designed by microwalk with ISIS 522247 Conjugate  IC₅₀ (μm) position IC₅₀ (nM) Hepato/ ISIS counted from HeLa/ Hepato/ Free  Hepato/ NO. 5′ 3′ Lipo Lipo up-take Electro  522247* N/A N/A 1.5 1.5 0.07 2 534714 21 1 1.5 2.5 2 5 534711 19 3 1.5 3 2 7 534713 11 11 >50 >10 ND 20 533814 8 14 0.8 1.5 0.1 1.5 533815 6 16 1 3 0.8 1.5 533813 4 18 1.8 5 >20 8 533816 1 21 1 2 >20 2

Example 20 Protein Binding in Mouse Plasma for C10-Conjugated Oligonucleotides

Several modified oligonucleotides from Table 6 were selected and evaluated for protein binding in mouse plasma. The protein binding of the oligonucleotides was measured by ultracentrifugation using the procedures as described herein.

The C10-conjugated oligomeric compounds were prepared as per the procedures illustrated in Example 17 and are described in Table 8. A subscript “s” between two nucleosides indicates a phosphorothioate internucleoside linkage (going 5′ to 3′). The absence of a subscript “s” between two nucleosides indicates a phosphodiester internucleoside linkage. A “Pv” at the 5′-end indicates a 5′-(E)-vinylphosphonate group, (PO(OH)₂(CH═CH—). A “Po” at the 5′-end indicates a 5′-phosphate group, (PO(OH)₂)—. Nucleosides followed by a subscript “f”, “m” or “e” are sugar modified nucleosides. A subscript “f” indicates a 2′-fluoro modified nucleoside, a subscript “m” indicates a 2′-O-methyl modified nucleoside and a subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside. ^(me)C indicates a 5-methyl cytosine nucleoside. Nucleosides with a subscript “C10” are shown below. Underlined nucleosides indicate the conjugate position. “NA” indicates not applicable.

Recovery Testing

Samples of oligonucleotides were prepared at 1 μM in PBS. Ultrafree-MC (Millipore) 30,000 NMWL filter units were placed in clean microcentrifuge tubes and pre-treated with 20 μL 0.5% Tween 80 in PBS and centrifuged for 10 minutes at 2,000 g. Pre-treated filters were transferred to the original collection tubes provided by Millipore. 300 μL of 1 μM of oligonucleotide was loaded onto the filters and immediately centrifuged for 20 min at 2,000 g. The concentrations in unfiltered solution and filtered solution were measured by Hybridization-Dependent ELISA using a complementary DNA probes conjugated with biotin and digoxigenin. The percent recovery was calculated using the formula provided: % Recovery=([pre-filtered]−[filtered])/[pre-filtered].

Capacity Study

Oligonucleotides were prepared at 1 μM in fresh CD-1 mouse plasma preserved with EDTA (K3) (BioChemed Services), mixed well, and kept on ice. Ultrafree-MC 30,000 NMWL spin filters (Millipore) were placed in clean microcentrifuge tubes and pre-treated with 20 μL 0.5% Tween 80 in PBS as described in recovery testing. Pre-treated spin filters were placed in collection tubes they were supplied with and 300 μL aliquots of 1 μM were then loaded onto spin filters in triplicate and incubated at 37° C. for 30 minutes. After incubation, samples were immediately centrifuged at 2,000 g for 20 minutes at room temperature (˜45 μL filtrate was collected). The concentration of oligonucleotide in unfiltered plasma and filtered plasma samples were determined by Hybridization-Dependent ELISA using complementary DNA probes conjugated with biotin and digoxigenin. The proportion of unbound oligonucleotide, adjusted for recovery was determined using the formulae provided: % Unbound=[filtered]/[unfiltered])  Formula 1. Adjust % Unbound=% Unbound/% Recovery  Formula 2. Protein Binding Analysis

The protein binding in mouse plasma for modified oligonucleotides was analyzed using the procedures as described above. Results in Table 8 are presented as Adjusted % Unbound and is denoted as “Unbound (%)”.

The parent oligonucleotide lacking a conjugate group, ISIS 522247, from which the newly designed oligonucleotides were derived from is marked with an asterisk (*) in the table. The 5-10-5 MOE gapmer, 104838 was also included in the study as a benchmark oligonucleotide against which the protein binding of the conjugated oligonucleotides could be compared.

As illustrated in Table 8, C10-conjugated oligonucleotides showed comparable protein binding in mouse plasma as compared to 5-10-5 MOE gapmer, ISIS 104838 while exhibiting a significant improvement in protein binding as compared to the non-conjugated parent oligonucleotide, ISIS 522247.

TABLE 8 Comparison of protein binding in mouse plasma of C10-conjugated oligonucleotides with ISIS 522247 and ISIS 104838 Conjugate position counted from Unbound SEQ ID ISIS NO. Composition (5′ to 3′) 5′ 3′ (%) NO. 104838 G_(es) ^(me)C_(es)T_(es)G_(es)A_(es)T_(s)T_(s)A_(s)G_(s)A_(s)G_(s)A_(s)G_(s)A_(s)G_(s)G_(es)T_(es) ^(me)C_(es) ^(me)C_(es) ^(me)C_(e) N/A N/A 1.76 21 (5-10-5) MOE gapmer  522247* Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs)U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) N/A N/A 7.06 6 533814 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m) T_(C10s) A_(m)A_(fs)U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) 8 14 3.32 22 533815 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m) T_(C10s) A_(m)U_(fs)A_(m)A_(fs)U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) 6 16 1.68 23 533816 Po-T_(C10s) U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs)U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) 1 21 1.38 6

Example 21 Modified Oligonucleotides Comprising Various Conjugates at Various Positions

A series of conjugated oligonucleotides comprising a 5′-(E)-vinylphosphonate or a 5′-phosphate group were designed based on the parent oligomeric compound, ISIS 522247 from Table 2 and is marked with an asterisk (*) in the table. The modified oligonucleotides were designed by introducing C16, C22 or cholesterol conjugate group at various positions of the oligonucleotides in an effort to determine the effects of conjugate groups on potency in reducing PTEN mRNA levels.

The conjugated oligonucleotides were prepared as per the procedures illustrated in Example 17 and are described in Table 9. A subscript “s” between two nucleosides indicates a phosphorothioate internucleoside linkage (going 5′ to 3′). The absence of a subscript “s” between two nucleosides indicates a phosphodiester internucleoside linkage. A “Pv” at the 5′-end indicates a 5′-(E)-vinylphosphonate group, (PO(OH)₂(CH═CH—). A “Po” at the 5′-end indicates a 5′-phosphate group, (PO(OH)₂—). Nucleosides followed by a subscript “f”, “m” or “e” are sugar modified nucleosides. A subscript “f” indicates a 2′-fluoro modified nucleoside, a subscript “m” indicates a 2′-O-methyl modified nucleoside and a subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside. Nucleosides with a subscript “C16”, “C22” or “Cholesterol” are shown below. Underlined nucleosides indicate the conjugate position. “NA” indicates not applicable.

TABLE 9 Modified oligonucleotides comprising C16, C22 or cholesterol conjugate Conjugate position SEQ ISIS counted from ID NO. Composition (5′ to 3′) Conjugate 5′ 3′ NO.  522247* Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs)U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) No N/A N/A  6 conjugate 543911 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)T _(C16s) A_(m)A_(fs)U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) C16 8 14 18 543912 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)T _(C16s) A_(m)U_(fs)A_(m)A_(fs)U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) C16 6 16 19 549166 Po-T _(C16s) U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs)U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) C16 1 21 14 551906 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)T _(C22s) A_(m)A_(fs)U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) C22 8 14 18 551907 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)T _(C22s) A_(m)U_(fs)A_(m)A_(fs)U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) C22 6 16 19 551908 Po-T _(C22s) U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs)U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) C22 1 21 14 543913 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m) T_(Cholesterols) A_(m)A_(fs)U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) Cholesterol 8 14 22 543914 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m) T_(Cholesterols) A_(m)U_(fs)A_(m)A_(fs)U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) Cholesterol 6 16 23 550959 Po-T_(cholesterols) U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs)U_(m)G_(fs)A_(m)U_(fs)C_(m)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) Cholesterol 1 21  6

Example 22 Modified Oligonucleotides Comprising Cholesterol Conjugate Targeting PTEN In Vitro Study

The conjugated oligonucleotide, ISIS 543913 from Table 8 comprising a 5′-(E)-vinylphosphonate and cholesterol conjugate at position 8, as counted from the 5′-terminus or position 14, as counted from the 3′-terminus was selected and evaluated for inhibition of PTEN mRNA levels.

The cholesterol-conjugated oligonucleotide was tested in vitro. Hepatocytes were treated with modified oligonucleotides using LIPOFECTAMINE™ 2000 (Lipo) as transfection method as described herein. The IC₅₀'s were calculated using the linear regression equation generated by plotting the normalized mRNA levels to the log of the concentrations used and the results are presented in Table 10.

The parent oligomeric compound, ISIS 522247, from which the newly designed oligonucleotide was derived from is marked with an asterisk (*) in the table and was included in the study as a benchmark oligonucleotide against which potency of the cholesterol-conjugated oligonucleotides could be compared. “NA” indicates not applicable.

As illustrated in Table 10, the modified oligonucleotide comprising a cholesterol conjugate at position 8, as counted from the 5′-terminus or position 14, as counted from the 3′-terminus exhibited comparable potency as compared to the parent oligonucleotide, ISIS 522247.

TABLE 10 Comparison of inhibition of PTEN mRNA levels of cholesterol-conjugated oligonucleotides in hepatocytes with ISIS 522247 Conjugate position IC₅₀ counted from SEQ ISIS NO. Composition (5′ to 3′) (μM) Conjugate 5′ 3′ ID NO. 522247* Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs) 1 No N/A N/A  6 U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) conjugate 543913  Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m) T_(Cholesterols) 3 Cholesterol 8 14 22 A_(m)A_(fs)U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e)

Example 23 Modified Oligonucleotides Comprising Various Conjugates Targeting PTEN In Vivo Single Dose Response Study

The modified oligonucleotides from Table 8 comprising a 5′-(E)-vinylphosphonate and various conjugates at position 8, as counted from the 5′-terminus or position 14, as counted from the 3′-terminus, were selected and evaluated for inhibition of PTEN mRNA levels in vivo.

Six week old BALB/C mice (Jackson Laboratory, Bar Harbor, Me.) were injected intravenously with modified oligonucleotides once a day at dosage 10, 50 or 100 mg/kg for one day. The mice were sacrificed 48 hrs following last administration. Liver tissues were homogenized and mRNA levels were quantitated using real-time PCR and normalized to Cyclophilin as described herein for comparison to untreated control levels (% UTC). The results are listed as the average % of PTEN mRNA expression for each treatment group relative to saline-injected control.

The parent oligonucleotide, ISIS 522247, from which the newly designed oligonucleotides was derived from is marked with an asterisk (*) in the table and was included in the study as a benchmark oligonucleotide against which potency of the conjugated oligonucleotides could be compared.

As illustrated in Table 11, treatment with modified oligonucleotides comprising a C16- or C22-conjugate at position 8, as counted from the 5′-terminus, or position 14, as counted from the 3′-terminus exhibited a significant increase in potency at 50 mg/kg or 100 mg/kg (ISIS 543911 and ISIS 551906) while the cholesterol-conjugate (ISIS 543913) showed comparable potency comparing to the parent oligonucleotide (ISIS 522247). The data presented in Table 11 demonstrated that inhibition of PTEN mRNA levels can be achieved with conjugated oligonucleotides.

TABLE 11 Comparison of PTEN mRNA levels of conjugated oligonucleotides with ISIS 522247 in vivo Conjugate position Dosage UTC counted from SEQ ID ISIS NO (mg/kg) (%) Conjugate 5' 3' NO 522247* 10 99 No N/A N/A 6 50 87.9 conjugate 100 61.2 543911 10 88.3 C16 8 14 18 50 35.6 100 28.1 551906 10 93.7 C22 8 14 18 50 40.9 543913 10 99.4 Cholesterol 8 14 22 100 67.2

Example 24 Modified Oligonucleotides Comprising Various Conjugates Targeting PTEN In Vivo Multiple Dose Study

Several modified oligonucleotides from Table 8 comprising a 5′-(E)-vinylphosphonate and various conjugates at position 8, as counted from the 5′-terminus or position 14, as counted from the 3′-terminus, were selected and evaluated for inhibition of PTEN mRNA levels in vivo.

Six week old BALB/C mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously with the modified oligonucleotides twice a day at dosage 25 mg/kg (100 mg/kg total) for two days. The mice were sacrificed 48 hrs following last administration. Liver tissues were homogenized and mRNA levels were quantitated using real-time PCR and normalized to Cyclophilin as described herein for comparison to untreated control levels (% UTC). The results are listed as the average % of PTEN mRNA expression for each treatment group relative to saline-injected control.

The parent oligonucleotide, ISIS 522247, from which the newly designed oligonucleotides were derived from is marked with an asterisk (*) in the table and was included in the study as a benchmark oligonucleotide against which potency of the conjugated oligonucleotides could be compared.

As illustrated in Table 12, modified oligonucleotides comprising a C16- or C22-conjugate at position 8 as counted from the 5′ terminus or position 14, as counted from the 3′-terminus showed a significant increase in potency at 50 mg/kg or 100 mg/kg (ISIS 543911 and ISIS 551906) while the cholesterol-conjugate (ISIS 543913) showed no improvement in potency comparing to the parent oligonucleotide (ISIS 522247).

TABLE 12 Comparison of PTEN mRNA levels of conjugated oligonucleotides with ISIS 522247 in vivo Dosage Conjugate position (mg/kg counted from SEQ ID ISIS NO total) % UTC Conjugate 5' 3' NO 522247* 100 45.5 No N/A N/A 6 conjugate 543911 100 17.9 C16 8 14 18 551906 100 21.5 C22 8 14 18 543913 100 98.9 Cholesterol 8 14 22

Example 25 Evaluation of the Stability of Modified Oligonucleotides In Vivo Study

The stability of modified oligonucleotides can be evaluated in vivo using the procedures as described herein. Liver tissues were harvested and collected on ice from BALB/C mice treated with modified oligonucleotides. 100-200 mg samples were minced and homogenized in 400 μL homogenization buffer (20 mM Tris, pH 8, 20 mM EDTA, 0.1 M NaCl, 0.5% NP-40). A standard curve ranging from 1 μg-75 μg was prepared for each ssRNA in 500 μL aliquots of control liver homogenate (400 μg/mL) with 10 μg internal standard (SEQ ID NO: 24, Isis NO: 355868, a 27-mer, 2′-O-methoxyethyl-modified phosphorothioate oligonucleotide). Tissue homogenates were then extracted using phenol/chloroform and solid support phase extraction techniques as described below with 300 μL NH₄OH and 800 μL phenol/chloroform/isoamyl alcohol used in the phenol/chloroform extraction.

Phenol/Chloroform Extraction

Stability of modified oligonucleotides was evaluated at time points 0, 5, 10, 20, 30, 40 and 60 minutes, except for SEQ ID NO: 25, Isis NO: 408877 which was evaluated at time points 0, 15, 30, 60, 120 and 240 mins; and SEQ ID NO: 26, Isis NO: 409044, at time points 0, 0.5, 1, 2, 4, 8, and 18 hours. An internal standard (SEQ ID NO: 24, Isis NO: 355868, a 27-mer, 2′-O-methoxyethyl-modified phosphorothioate oligonucleotide) with final concentration of 2.5 μM was added to each sample prior to extraction. Samples were extracted with 70 μL of NH₄OH and 240 μL of phenol/chloroform/isoamyl alcohol (25:24:1). The supernatant was removed after centrifugation at 14000 rpm for 2 min. The remaining extractant was vortexed with an additional 500 μL of water and the aqueous layer was removed and combined with the supernatant after centrifugation at 14000 rpm for 2 minutes.

Solid Phase Extraction

Triethylammonium acetate solution at 1M (500 μL) was added to the supernatant. The aqueous layer of the mixture was loaded onto the pre-conditioned Biotage™ Phenyl Solid Phase Extraction Plate (SPE plate) after centrifugation at 9000 rpm for 20 minutes. The SPE plate was washed several times with water. The sample was then eluted with 1.5 mL of 1% TEA in 90% MeOH and filtered through the Protein Precipitation Plate (Phenomenex™). The eluent was evaporated to dryness and diluted to 200 μL with 50% quenching buffer (8 M urea, 50 mM EDTA) and water before sample injection.

LC-MS

An Agilent 1100 Series LC/MSD system was connected in-line to a mass spectrometer. Mass spectrometer was operated in the electrospray negative ionization mode. The nebulizer nitrogen gas was set at 325 psi and the drying nitrogen gas was set at 12 L/min. The drying temperature was 325° C. Samples (25 μL/well) were introduced via an auto sampler and reversed-phase chromatography was carried out with an) (Bridge OST C18 2.5 μm 2.1 mm×50 mm HPLC column using a flow rate of 300 μL/min at 55° C. The ion pair buffers consisted of A: 5 mM tributylammonium acetate (TBAA) in 20% acetonitrile and B: 5 mM TBAA in 90% acetonitrile and the loading buffer was 25 mM TBAA in 25% Acetonitrile. Separation was performed on a 30% to 70% B in 9 min and then 80% B in 11 min gradient.

Quantitative analysis of oligonucleotide and internal standard by extracted ion chromatograms of the most abundant ions was performed using MSD ChemStation software.

The internal standard oligonucleotides are described in Table 12a. A subscript “s” between two nucleosides indicates a phosphorothioate internucleoside linkage (from the 5′ to the 3′ end). The absence of a subscript “s” between two nucleosides indicates a phosphodiester internucleoside linkage. A “Po” at the 5′-end indicates a 5′-phosphate group, (PO(OH)₂—). Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “f”, “m” or “e” are sugar modified nucleosides. A subscript “f” indicates a 2′-fluoro modified nucleoside, a subscript “m” indicates a 2′-O-methyl modified nucleoside and a subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside. ^(me)C indicates a 5-methyl cytosine nucleoside.

TABLE 12a Internal standard oligonucleotides SEQ ISIS NO Sequence (5′ to 3′) 5′-Chemistry ID NO 355868 G_(es) ^(me)C_(es)G_(s)T_(s)T_(s)T_(s)G_(s)C_(s)T_(s)C_(s)T_(s)T_(s) OH 24 C_(s)T_(s)T_(s) ^(me)C_(es)T_(es)T_(es)G_(es) ^(me)C_(es)G_(es)T_(s) T_(s)T_(s)T_(s)T_(s)T_(e) 408877 Po-U_(m)U_(f)G_(f)U_(f)C_(f)U_(m)C_(m)U_(f)G_(f)G_(ms)U_(ms) Phosphate 25 C_(fs)C_(fs)U_(fs)U_(fs)A_(fs)C_(ms)U_(ms)U_(ms)T_(es)T_(e) 409044 Po-U_(ms)U_(f)G_(m)U_(f)C_(m)U_(f)C_(m)U_(f)G_(m)G_(f)U_(m)C_(f) Phosphate 26 C_(m)U_(fs)U_(ms)A_(fs)C_(ms)U_(fs)U_(ms)A_(es)A_(e)

Example 26 Modified Oligonucleotides Comprising C10-Conjugate Targeting PTEN In Vivo Multiple Dose Response and Stability Studies

The modified antisense oligonucleotide, ISIS 533814 from Table 6 comprising a 5′-(E)-vinylphosphonate and a C-10 conjugate group at position 8, as counted from the 5′-terminus or position 14, as counted from the 3′-terminus, was selected and evaluated for inhibition of PTEN mRNA levels in vivo.

Six week old BALB/C mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously twice a day at dosage 25 mg/kg (50, 100, or 200 mg/kg total) for one, two or four days with C10-conjugated oligonucleotide, ISIS 533814 or twice a day at dosage 25 mg/kg (100 mg/kg total) for two days with the parent oligonucleotide, ISIS 522247 or the 5-10-5 MOE gapmer, ISIS 116847. The mice were sacrificed 48 hrs following last administration. Liver tissues were homogenized and mRNA levels were quantitated using real-time PCR and normalized to Cyclophilin as described herein for comparison to untreated control levels (% UTC). The results in Table 13 are listed as the average % of PTEN mRNA expression for each treatment group relative to saline-injected control.

The parent oligonucleotide lacking a conjugate group, ISIS 522247, from which the newly designed oligonucleotides were derived from is marked with an asterisk (*) in the table. The 5-10-5 MOE gapmer is described in Table 4 and was also included in the study as a benchmark oligonucleotide against which potency of the conjugated oligonucleotides could be compared. “NA” indicates not applicable.

As illustrated in Table 13, modified oligonucleotides comprising a C10-conjugate at position 8, as counted from the 5′-terminus, or position 14, as counted from the 3′-terminus showed reduction in PTEN mRNA levels in a similar manner as the parent oligonucleotide, ISIS 522247.

The modified oligonucleotides were also evaluated for in vivo stability at dosage 100 mg/kg total and the tissue samples were collected and prepared using the same technique described in Example 25. Quantitative analysis of the oligonucleotides standard were performed by extracted ion chromatograms in the most abundant charge state (−4) using Chemstation software. The liver concentration (μg/g) of full length oligonucleotides comprising a 5′-terminal phosphonate group was measured by LC/MS and the results are presented below.

As illustrated in Table 14 and in FIG. 1, similar concentration of full-length oligonucleotide was observed for C10-conjugate (ISIS 533814) in liver accumulation as compared to the parent oligonucleotide, ISIS 522247. The full-length oligonucleotide for ISIS 533814 lacks a C10 conjugate due to hydrolysis at the amide bond between the alkyl linker group and the conjugate.

TABLE 13 Comparison of PTEN mRNA levels of C10-conjugated oligonucleotides with ISIS 116847 and ISIS 522247 in multiple dose response study in vivo Dosage Conjugate position SEQ (mg/kg UTC Conju- counted from ID ISIS NO. Day(s) total) (%) gate 5' 3' NO. 116847 2 100 12.7 No N/A N/A 8 (5-10-5) conjugate MOE gapmer 522247* 2 100 43.9 No N/A N/A 6 conjugate 533814 1 50 70.1 C10 8 14 18 2 100 44.4 4 200 38.0

TABLE 14 Comparison of full-length C10-conjugated oligonucleotides in liver accumulation with ISIS 522247 in vivo Liver conc. of full Conjugate/ Dosage length ssRNA position ISIS NO. Day(s) (mg/kg total) (μg/g) counted from 5′ 116847 2 100 151.62 ± 29.2  (5-10-5) MOE gapmer 522247* 2 100 98.22 ± 3   No conjugate 533814 1 50 208.61 ± 45.8  C10, pos 8 2 100 95.4 ± 9.4 C10 conjugate 4 200 60.88 ± 22.9 hydrolyzed

Example 27 Modified Oligonucleotides Comprising C16 Conjugate at the 3′-Terminus Targeting PTEN—In Vivo Multiple Dose Response Study

The modified oligonucleotides from Table 4 comprising a 5′-(E)-vinylphosphonate and C16 conjugate at the 3′-terminus were selected and evaluated for inhibition of PTEN mRNA levels in vivo.

Six week old BALB/C mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously with modified oligonucleotides twice a day at dosage 25 mg/kg (50 or 100 mg/kg total) for one or two days. The mice were sacrificed 48 hrs following last administration. Liver tissues were homogenized and mRNA levels were quantitated using real-time PCR and normalized to Cyclophilin as described herein for comparison to untreated control levels (% UTC). The results in Table 15 are listed as the average % of PTEN mRNA expression for each treatment group relative to saline-injected control.

The parent oligonucleotide lacking a conjugate group, ISIS 522247, from which the newly designed oligonucleotides were derived from is marked with an asterisk (*) in the table. The 5-10-5 MOE gapmer, ISIS 116847 was also included in the study as a benchmark oligonucleotide against which potency of the conjugated oligonucleotides could be compared. “NA” indicates not applicable.

As illustrated in Table 15, the modified oligonucleotide comprising a C16-conjugate at the 3′-terminus showed reduction in PTEN mRNA levels in a similar manner as the parent oligonucleotide, ISIS 522247.

TABLE 15 Comparison of PTEN mRNA levels of conjugated oligonucleotides with ISIS 116847 and ISIS 522247 in multiple dose response study in vivo Dosage Conjugate position SEQ (mg/kg UTC counted from ID ISIS NO Day(s) total) (%) Conjugate 5' 3' NO 116847 1 50 42.9 No N/A N/A 8 (5-10-5) conjugate MOE gapmer 522247* 1 50 74.9 No N/A N/A 6 2 100 47.1 conjugate 526608 1 50 95.0 C16 21 1 6 2 100 69.7

Example 28 Relative Distribution of 5′-(E)-Vinyl Phosphonate ssRNA Lipid Conjugates in Mouse Plasma

Oligonucleotides, Isis Nos. 116847, 52247, 533814, and 543911, were prepared in 50% mouse plasma (K3, EDTA) at the following concentrations (uM): 0.1, 0.5, 1, 5, 10, and 25. A PBS control was prepared at 1 uM. The oligonucleotides in plasma or PBS were then incubated at 37° C. on an HPLC autosampler for at least 30 minutes. 100 uL samples were then separated by HPLC on a Zenix SEC300 (4.5×300 mm) size exclusion column using 0.35 mL/min PBS mobile phase. The column was kept at 10° C. during separation. Four 1.5 minute fractions were collected from 5-11 minutes. Fractions correspond with elution times of proteins with the approximate masses: 5-6.5 min=>300 kDa; 6.5-8 min=100-300 kDa; 8-9.5 min=30-100 kDa, and 9.5-11 min=<30 kDa, or unbound oligonucleotide. The concentration of the oligonucleotides in each fraction was determined by hybridization-dependent ELISA and the distribution of oligonucleotides across fractions was reported relative to total. The results of the assay are illustrated in Table 16. The results illustrate that oligonucleotides with C16 conjugates at position 8 from the 5′-end or position 14 from the 3′ end increase and alter binding to high molecular weight plasma proteins.

TABLE 16 Relative Distribution of 5'-(E)-Vinyl Phosphonate ssRNA Lipid Conjugates In Mouse Plasma Fractional Conjugate Position Concentration 5 % 10 % 25 % Counted From SEQ Isis No (μM) μM Bound μM Bound μM Bound 5' 3' ID NO. 116847 >300 kDa 0.1 2 0.1 1 0.2 0.8 N/A N/A 8 100-300 kDa 0.2 4 0.2 2 0.2 0.8 N/A N/A 30-100 kDa 1.3 26 1.4 14 1.5 6 N/A N/A >30 kDa 3.4 68 8.3 83 23.1 92.4 N/A N/A 52247 >300 kDa 0 0 0.1 1 0 0 N/A N/A 6 100-300 kDa 0.1 2 0.2 2 0.3 1.2 N/A N/A 30-100 kDa 1.9 38 2.3 23 2.6 10.4 N/A N/A >30 kDa 3 60 7.4 74 22.1 88.4 N/A N/A 533814 >300 kDa 0.3 6 0.4 4 0.6 2.4 8 14 18 (C₁₀) 100-300 kDa 0.7 14 0.8 8 1.3 5.2 8 14 30-100 kDa 1.9 38 2.5 25 2.2 8.8 8 14 >30 kDa 2 40 6.2 62 20.9 83.6 8 14 543911 >300 kDa 0.2 4 0.5 5 1.1 4.4 8 14 18 (C₁₆) 100-300 kDa 1.2 24 1.2 12 2.9 11.6 8 14 30-100 kDa 2.5 50 4.3 43 9.4 37.6 8 14 >30 kDa 1 20 4 40 11.5 46 8 14

Example 29 Preparation of Compound 10, 2-Acetamido-2-deoxy-D-galactopyranose conjugate

Compound 7 is prepared according to the procedures published in WO 2000/14048 or by Manoharan et al., J. Org. Chem., 1999, 64, 6468-6472. Compound 8 is prepared according to the procedures published by Rensen et al., J. Med. Chem., 2004, 47, 5798-5808.

Example 30 Preparation of Compound 14, Squalene Conjugate

Compound 7 is prepared according to the procedures published in WO 2000/14048 or by Manoharan et al., J. Org. Chem., 1999, 64, 6468-6472. Compound 11 is prepared using the procedures according to Raouane et al., J. Med. Chem., 2011, 54, 4067-4076.

Example 31 Preparation of Compounds 27-32

Compounds 15-20 are prepared as per the procedures illustrated in Examples 40 and 41. Compound 8 is prepared according to the procedures published by Rensen et al., J. Med. Chem., 2004, 47, 5798-5808.

Example 32 Preparation of Compounds 45-50

Compounds 33-38 are prepared using similar procedures as illustrated in Example 42. Compound 8 is prepared according to the procedures published by Rensen et al., J. Med. Chem., 2004, 47, 5798-5808.

Example 33 Preparation of Compounds 63-68

Compounds 51-56 are prepared using similar procedures as illustrated in Example 42. Compound 8 is prepared according to the procedures published by Rensen et al., J. Med. Chem., 2004, 47, 5798-5808.

Example 34 Preparation of Compound 71

Compound 69 is prepared using similar procedures as illustrated in Example 40. Compound 8 is prepared according to the procedures published by Rensen et al., J. Med. Chem., 2004, 47, 5798-5808.

Example 35 Preparation of Compound 74

Compound 72 is prepared using similar procedures as illustrated in Example 40. Compound 8 is prepared according to the procedures published by Rensen et al., J. Med. Chem., 2004, 47, 5798-5808.

Example 36 Preparation of Compound 76

Compound 75 is prepared as per the procedures illustrated in Example 17. Compound 8 is prepared according to the procedures published by Rensen et al., J. Med. Chem., 2004, 47, 5798-5808.

Example 37 Preparation of Compound 78

Compound 77 is prepared as per the procedures illustrated in Examples 17, 43 and 44. Compound 8 is prepared according to the procedures published by Rensen et al., J. Med. Chem., 2004, 47, 5798-5808.

Example 38 Preparation of Compound 80

Compound 79 is prepared as per the procedures illustrated in Examples 17, 43 and 44. Compound 8 is prepared according to the procedures published by Rensen et al., J. Med. Chem., 2004, 47, 5798-5808.

Example 39 Preparation of Compound 83

Compound 7 is prepared according to the procedures published in WO 2000/14048 or by Manoharan et al., J. Org. Chem., 1999, 64, 6468-6472. Compound 81 is prepared using similar procedures according to Rensen et al., J. Med. Chem., 2004, 47, 5798-5808.

Example 40 Preparation of Compound 85

Compound 84, N-Acetylneuraminic acid is commercially available from various sources.

Example 41 Preparation of Compound 15

Compound 86, 1,2-O-isopropylidene-α-D-ribofuranose is commercially available from various sources.

Example 42 Preparation of Compounds 16-20

Compound 89 is prepared as per the procedures illustrated in Example 40.

Example 43 Preparation of Compound 33

Compound 97 is commercially available from various sources.

Example 44 General Procedure for the Preparation of Compound 103

Compound 99 is commercially available from various sources. Compound 100 is prepared according to the procedures published by Dellinger et al., J. Am. Chem. Soc., 2003, 125, 940-950.

Example 45 General Procedure for the Preparation of Compound 106

The Unylinker™ 104 is commercially available. Conjugated oligomeric Compound 103 is prepared as per the procedures illustrated in Example 43.

Example 46 Modified Oligonucleotides Comprising C10, C16 or C22 Conjugate at Position 1 or 8 Targeting PTEN In Vitro Study

Several conjugated oligonucleotides from Tables 5 and 9 were selected and evaluated for their effects on PTEN mRNA expression level in vitro. These oligonucleotides were designed by introducing a C10, C16 or C22 conjugate at position 1 or 8, as counted from the 5′-terminus. ISIS 522247 lacking a conjugate group was included in the study for comparison.

The conjugated modified oligonucleotides were tested in vitro using two transfection methods. Hepatocytes at a density of 12,000 cells per well were transfected using LIPOFECTAMINE™ 2000 (Lipo) with 0.27, 0.82, 2.46, 7.40, 22.2, 66.67 and 200 nM concentrations or at a density of 35,000 cells per well using electroporation (Electro) with 0.027, 0.08, 0.25, 0.74, 2.20, 6.67 and 20 μM concentrations of conjugated oligonucleotides. After a treatment period of approximately 24 hours, RNA was isolated from the cells and PTEN mRNA levels were measured by quantitative real-time PCR. Primer probe set RTS186 was used to measure mRNA levels. PTEN mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is presented in Table 17 and was calculated by plotting the concentrations of oligonucleotides used versus the percent inhibition of PTEN mRNA expression achieved at each concentration, and noting the concentration of oligonucleotide at which 50% inhibition of PTEN mRNA expression was achieved compared to the control. As illustrated in Table 17, the oligonucleotide with a 5′-phosphate and a C16 conjugate at position 1 (ISIS 549166) showed comparable potency in inhibiting PTEN mRNA levels as ISIS 522247 by both transfection methods.

TABLE 17 Comparison of inhibition of PTEN mRNA levels of C10, C16 or C22 conjugated oligonucleotides with ISIS 522247 Conjugate position Hepato/Lipo Heptato/Electro ISIS NO. Conjugate counted from 5′ 5′-Chemistry IC₅₀ (nM) IC₅₀ (μM) 522247 No conjugate N/A (E)—(PO(OH)₂(CH═CH)— 1.5 2 549621 C10 1 (E)—(PO(OH)₂(CH═CH)— ND 1.5 549166 C16 1 (PO(OH)₂)— 1.2 2 551908 C22 1 (PO(OH)₂)— 8 15 543911 C16 8 (E)—(PO(OH)₂(CH═CH)— 1.2 20 551906 C22 8 (E)—(PO(OH)₂(CH═CH)— 8 >20 N/A = Not Applicable; ND = no data

Example 47 Modified Oligonucleotides Comprising C16 Conjugate at Various Positions Targeting PTEN In Vitro Study

A series of conjugated oligonucleotides was designed based on the parent oligomeric compound lacking the conjugate group, ISIS 522247. The conjugated oligonucleotides comprising a 5′-(E)-vinylphosphonate were created with a C16 conjugate group shifted slightly upstream or downstream (i.e. “microwalk’) of the oligonucleotide. The newly designed conjugated oligonucleotides were tested and evaluated for their effects on PTEN mRNA levels in vitro. ISIS 522247 was included in the study for comparison.

The conjugated oligonucleotides were prepared using similar procedures as illustrated in Examples 12 and 17 and are described in Table 18. A subscript “s” between two nucleosides indicates a phosphorothioate internucleoside linkage (going 5′ to 3′). The absence of a subscript “s” between two nucleosides indicates a phosphodiester internucleoside linkage. A “Pv” at the 5′-end indicates a 5′-(E)-vinylphosphonate group, (PO(OH)₂(CH═CH—). Nucleosides followed by a subscript “f”, “m” or “e” are sugar modified nucleosides. A subscript “f” indicates a 2′-fluoro modified nucleoside, a subscript “m” indicates a 2′-O-methyl modified nucleoside and a subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside. Nucleosides with a subscript “C16” are shown below. Underlined nucleosides indicate the conjugate position.

The conjugated modified oligonucleotides were tested in vitro using two transfection methods. Hepatocytes at a density of 12,000 cells per well were transfected using LIPOFECTAMINE™ 2000 (Lipo) with 0.27, 0.82, 2.46, 7.40, 22.2, 66.67 and 200 nM concentrations or at a density of 35,000 cells per well using electroporation (Electro) with 0.027, 0.08, 0.25, 0.74, 2.20, 6.67 and 20 μM concentrations of conjugated oligonucleotides. After a treatment period of approximately 24 hours, RNA was isolated from the cells and PTEN mRNA levels were measured by quantitative real-time PCR. Primer probe set RTS186 was used to measure mRNA levels. PTEN mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.

The IC₅₀ of each oligonucleotide was calculated using the method described previously and the results are presented in Table 19. As illustrated, all but one conjugated oligonucleotides showed comparable potency in inhibiting PTEN mRNA levels by Lipofectamine as compared to ISIS 522247 lacking the conjugate.

TABLE 18 Modified oligonucleotides comprising C16 conjugate C16 conjugate position SEQ ISIS NO. Composition (5′ to 3′) counted from 5′-terminus ID NO. 522247 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs) No conjugate 6 U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) 580933 Pv-T_(C16s) U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs)  1 6 U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) 576803 Pv-T_(es)U_(fs)A_(m)U _(C16s) C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs)  4 6 U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) 576798 Pv-T_(es)U_(fs)A_(m)U_(fs) C_(C16) U_(fs)A_(m)U_(fs)A_(m)A_(fs)  5 6 U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) 576797 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m) U_(C16s) A_(m)U_(fs)A_(m)A_(fs)  6 6 U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) 571032 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m) U_(C16s) A_(m)A_(fs)  8 6 U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) 576796 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs) 14 6 U_(m)G_(fs)A_(m) U_(C16s) C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) 579694 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs) 15 6 U_(m)G_(fs)A_(m)U_(fs) C_(C16s) A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) 576794 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs) 19 6 U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs) U_(C16s) A_(es)A_(e) 576793 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs) 20 6 U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms) U_(C16s) A_(e) 576792 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs) 21 6 U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es) U_(C16)

TABLE 19 Comparison of inhibition of PTEN mRNA levels of C16-conjugated oligonucleotides with ISIS 522247 Lipo Electro SEQ ISIS IC₅₀ IC₅₀ C16 conjugate position ID NO. (nM) (μM) counted from 5′-terminus NO 522247 1 1 No conjugate 6 580933 1.5 4  1 6 576803 1.5 10  4 6 576798 1 8  5 6 576797 1 8  6 6 571032 1 >20  8 6 576796 7 >20 14 6 579694 0.9 10 15 6 576794 0.9 6 19 6 576793 1.5 >20 20 6 576792 0.9 7 21 6

Example 48 Modified Oligonucleotides Comprising Hexylamino (HA) at Position 1 or 8 Targeting PTEN In Vitro Study

Additional conjugated oligonucleotides were designed based on the parent oligomeric compound lacking the conjugate group, ISIS 522247. The conjugated oligonucleotides were designed by introducing a hexylamino (HA) or C16 conjugate group at position 1 or 8 of the oligonucleotide. The newly designed conjugated oligonucleotides were tested and evaluated for their effects on PTEN mRNA levels in vitro. ISIS 522247 was included in the study for comparison.

The conjugated oligonucleotides were prepared using similar procedures as illustrated in Examples 12 and 17 and are described in Table 20. A subscript “s” between two nucleosides indicates a phosphorothioate internucleoside linkage (going 5′ to 3′). The absence of a subscript “s” between two nucleosides indicates a phosphodiester internucleoside linkage. A “Pv” at the 5′-end indicates a 5′-(E)-vinylphosphonate group, (PO(OH)₂(CH═CH—). A “Po” at the 5′-end indicates a 5′-phosphate group, (PO(OH)₂—). Nucleosides followed by a subscript “1”, “m” or “e” are sugar modified nucleosides. A subscript “f” indicates a 2′-fluoro modified nucleoside, a subscript “m” indicates a 2′-O-methyl modified nucleoside and a subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside. Nucleosides with a subscript “C16” or HA are shown below. Underlined nucleosides indicate the conjugate position.

The conjugated modified oligonucleotides were tested in vitro using two transfection methods. Hepatocytes at a density of 12,000 cells per well were transfected using LIPOFECTAMINE™ 2000 (Lipo) with 0.27, 0.82, 2.46, 7.40, 22.2, 66.67 and 200 nM concentrations or at a density of 35,000 cells per well using electroporation (Electro) with 0.027, 0.08, 0.25, 0.74, 2.20, 6.67 and 20 μM concentrations of conjugated oligonucleotides. After a treatment period of approximately 24 hours, RNA was isolated from the cells and PTEN mRNA levels were measured by quantitative real-time PCR. Primer probe set RTS186 was used to measure mRNA levels. PTEN mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.

The IC₅₀ of each oligonucleotide was calculated using the method described previously and the results are presented in Tables 21 and 22. As illustrated, incorporation of hexylamino at position 1 (ISIS 582081) or 8 (ISIS 576459) showed comparable potency in inhibiting PTEN mRNA levels while C16 conjugated oligonucleotides showed reduction in potency as compared to the parent compound, ISIS 522247.

TABLE 20 Modified oligonucleotides comprising hexylamino (HA) or C16 conjugate Conjugate position counted SEQ ISIS NO Composition (5′ to 3′) Conjugate from 5′ 5′-Chemistry ID NO. 522247 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs) No N/A (E)-(PO(OH)₂(CH═CH)— 6 U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) conjugate 580933 Pv-T_(C16s) U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs)U_(m) C16 1 (E)-(PO(OH)₂(CH═CH)— 6 G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) 582081 Po-U_(HAs) U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs)U_(m) HA 1 (PO(OH)₂- 6 G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) 571032 Pv-T_(s)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m) U_(C16s) A_(m)A_(fs)U_(m) C16 8 (E)-(PO(OH)₂(CH═CH)— 6 G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) 576459 Pv-T_(s)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m) U_(HAs) A_(m)A_(fs)U_(m) HA 8 (E)-(PO(OH)₂(CH═CH)— 6 G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) N/A = Not Applicable

TABLE 21 Comparison of inhibition of PTEN mRNA levels of hexylamino (HA) or C16 conjugated oligonucleotides at position 1 with ISIS 522247 Conjugate position IC₅₀ ISIS NO. Conjugate counted from 5′ 5′-Chemistry (μM) 522247 No conjugate N/A (E)-(PO(OH)₂(CH═CH)— 0.57 580933 C16 1 (E)-(PO(OH)₂(CH═CH)— 3.3 582081 HA 1 (PO(OH)₂- 0.94

TABLE 22 Comparison of inhibition of PTEN mRNA levels of hexylamino (HA) or C16 conjugated oligonucleotides at position 8 with ISIS 522247 Conjugate position ISIS Conju- counted IC₅₀ NO. gate from 5′ 5′-Chemistry (μM) 522247 No N/A (E)-(PO(OH₂(CH═CH)— 0.82 conju- gate 571032 C16 8 (E)-(PO(OH₂(CH═CH)—  7.8 576459 HA 8 (E)-(PO(OH₂(CH═CH)—  1.1 N/A = Not Applicable

Example 49 Modified Oligonucleotides Comprising C16 Conjugate Targeting PTEN In Vivo Study

ISIS 543911 comprising a 5′-(E)-vinylphosphonate and C16 conjugate at position 8, as counted from the 5′-terminus was selected from the previous examples and evaluated for inhibition of PTEN mRNA levels in vivo. ISIS 522247 lacking a conjugate group and 5-10-5 MOE gapmer, ISIS 116847 were included in the study for comparison.

Six week old BALB/C mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once a day for one day with ISIS 116847 or 543911 at 3, 10 and 30 mg/kg or with saline treated control. Another group of mice was injected subcutaneously twice a day for two days at 25 mg/kg (100 mg/kg total) with ISIS 116847, 522247 or 543911. Each treatment group consisted of 4 animals. The mice were sacrificed 48 hrs following last administration. Liver tissues were homogenized and mRNA levels were quantitated using real-time PCR and normalized to RIBOGREEN as described herein. The results in Table 23 are presented as percent of PTEN mRNA expression, relative to untreated control levels and is denoted as “% UTC”. As illustrated, inhibition of PTEN mRNA levels was achieved in a dose-dependent manner with the conjugated oligonucleotide. In addition, ISIS 543911 comprising a C16 conjugate at position 8 showed an increase in potency at 100 mg/kg as compared to the parent compound lacking a conjugate group, ISIS 522247.

TABLE 23 Comparison of inhibition of PTEN mRNA levels of C16 conjugated oligonucleotides at position 8 with ISIS 522247 and 5-10-5 MOE gapmer, ISIS 116847 Dosage Conjugate/position ISIS NO (mg/kg) % UTC counted from 5′ Saline 0 100 N/A 116847 3 95.77 5-10-5 10 76.48 MOE gapmer 30 44.74 100 21.46 100 (25 mg/kg twice 15.63 a day for 2 days) 522247 100 (25 mg/kg twice 45.59 No conjugate a day for 2 days) 543911 3 92.79 C16, pos 8 10 86.84 30 72.73 100 (25 mg/kg twice 24.42 a day for 2 days) N/A = Not Applicable

Example 50 Modified Oligonucleotides Comprising C16 Conjugate Targeting PTEN In Vivo Study

ISIS 571032 comprising a 5′-(E)-vinylphosphonate and C16 conjugate at position 8, as counted from the 5′-terminus was selected from the previous examples and evaluated for inhibition of PTEN mRNA levels in vivo. ISIS 522247 lacking a conjugate group and 5-10-5 MOE gapmer, ISIS 116847 were included in the study for comparison.

Six week old BALB/C mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once a day for one day with ISIS 116847 at 10, 30 and 100 mg/kg or 571032 at 7, 18, 44 and 110 mg/kg or with saline treated control. Another group of mice was injected subcutaneously twice a day for two days at 25 mg/kg (100 mg/kg total) with ISIS 522247. Each treatment group consisted of 4 animals. The mice were sacrificed 48 hrs following last administration. Liver tissues were homogenized and mRNA levels were quantitated using real-time PCR and normalized to GAPDH. The results in Table 24 are presented as percent of PTEN mRNA expression, relative to untreated control levels and is denoted as “% UTC”. As illustrated, inhibition of PTEN mRNA levels was achieved in a dose-dependent manner with the conjugated oligonucleotide.

TABLE 24 Comparison of inhibition of PTEN mRNA levels of C16 conjugated oligonucleotide with ISIS 522247 and 5-10-5 MOE gapmer, ISIS 116847 Dosage Conjugate/position ISIS NO (mg/kg) % UTC counted from 5′ Saline 0 100 N/A 116847 10 83.48 5-10-5 30 35.22 MOE gapmer 100 15.20 522247 100 (25 mg/kg twice 59.12 No conjugate a day for 2 days) 571032 7 82.05 C16, pos 8 18 73.32 44 54.74 110 32.95 N/A = Not Applicable

Example 51 Modified Oligonucleotide Comprising C16 Conjugate Targeting PTEN In Vivo Multiple Dose Response Study

ISIS 571032 comprising a 5′-(E)-vinylphosphonate and C16 conjugate at position 8, as counted from the 5′-terminus was selected from the previous example and evaluated for multiple dose response study in vivo targeting PTEN. ISIS 522247 lacking a conjugate group and 5-10-5 MOE gapmer, ISIS 116847 were included in the study for comparison.

Six week old BALB/C mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously twice a week for three weeks with the conjugated oligonucleotide at dosage presented in Table 24 or with saline treated control. One group of mice was injected subcutaneously twice a day for two days at 25 mg/kg (100 mg/kg/wk total) with ISIS 522247. Each treatment group consisted of 4 animals. The mice were sacrificed 48 hrs following last administration. Liver tissues were homogenized and mRNA levels were quantitated using real-time PCR and normalized to Cyclophilin as described herein. The results in Table 25 are presented as percent of PTEN mRNA expression, relative to untreated control levels and is denoted as “% UTC”. As illustrated, inhibition of PTEN mRNA levels was achieved in a dose-dependent manner with the conjugated oligonucleotide.

TABLE 25 Comparison of inhibition of PTEN mRNA levels of C16 conjugated oligonucleotide with ISIS 522247 and 5-10-5 MOE gapmer, ISIS 116847 Dosage Conjugate/position ISIS NO (mg/kg/wk total) % UTC counted from 5′ Saline 0 100 — 116847 3 60.44 5-10-5 10 35.90 MOE gapmer 30 17.90 522247 100 49.35 No conjugate 571032 3 100.32 C16, pos 8 7 84.30 18 67.76 44 32.00

Example 52 Modified Oligonucleotide Comprising C16 Conjugate Targeting PTEN In Vivo Multiple Dose Response Study

ISIS 571032 comprising a 5′-(E)-vinylphosphonate and C16 conjugate at position 8, as counted from the 5′-terminus was selected from the previous example and evaluated for multiple dose response study in vivo targeting PTEN. ISIS 116847, a 5-10-5 MOE gapmer was included in the study for comparison.

Six week old BALB/C mice (Jackson Laboratory, Bar Harbor, Me.) were injected intravenously twice a week for three weeks with ISIS 116847 or 571032 at the dosage presented in Table 26 or with saline treated control. Each treatment group consisted of 4 animals. The mice were sacrificed 48 hrs following last administration. Liver tissues were homogenized and mRNA levels were quantitated using real-time PCR and normalized to Cyclophilin as described herein. The results in Table 26 are presented as percent of PTEN mRNA expression, relative to untreated control levels and is denoted as “% UTC”. As illustrated, inhibition of PTEN mRNA levels was achieved in a dose-dependent manner with the conjugated oligonucleotide.

TABLE 26 Comparison of inhibition of PTEN mRNA levels of C16 conjugated oligonucleotide with ISIS 522247 and 5-10-5 MOE gapmer, ISIS 116847 Dosage Conjugate/position ISIS NO (mg/kg/wk) % UTC counted from 5′ Saline 0 100 — 116847 6 79.21 5-10-5 20 42.45 MOE gapmer 60 21.67 571032 6 93.43 C16, pos 8 14 81.55 36 48.79 88 23.44

Example 53 Modified Oligonucleotide Comprising C16 Conjugate Targeting PTEN In Vivo Study

ISIS 571032 comprising a 5′-(E)-vinylphosphonate and C16 conjugate at position 8, as counted from the 5′-terminus was selected from the previous example and further evaluated for its effect in PTEN mRNA level in adipose tissue in vivo.

Six week old BALB/C mice (Jackson Laboratory, Bar Harbor, Me.) were treated twice a week for three weeks with ISIS 571032 at 44 mg/kg (88 mg/kg/wk total) by tail vein injection or with saline treated control. Each treatment group consisted of 4 animals. The mice were sacrificed 48 hrs following last administration. Liver tissues were homogenized and mRNA levels were quantitated using real-time PCR and normalized to Cyclophilin as described herein. The results in Table 27 are presented as the average percent of PTEN mRNA levels for each treatment group, normalized to PBS-treated control and is denoted as “% UTC”. As illustrated, inhibition of PTEN mRNA level in adipose tissue was achieved with the conjugated oligonucleotide.

TABLE 27 Effect of C16 conjugated oligonucleotide on PTEN mRNA level in adipose tissue Dosage Conjugate/position ISIS NO (mg/kg/wk) % UTC counted from 5′ Saline 0 100 — 571032 88 41.19 C16

Example 54 Modified Oligonucleotide Comprising C16 Conjugate Targeting PTEN In Vivo Study

ISIS 571032 and 580933 comprising a 5′-(E)-vinylphosphonate and C16 conjugate at position 8 or 1, as counted from the 5′-terminus was selected from previous examples and evaluated for their effect on PTEN mRNA in vivo.

Six week old BALB/C mice (Jackson Laboratory, Bar Harbor, Me.) were treated subcutaneously with ISIS 571032 at 27.5 mg/kg twice a day for two days (110 mg/kg total). One group of mice was treated with ISIS 580933 at 2.8, 7, 18 and 44 mg/kg once a day for one day and 27.5 mg/kg twice a day for two days (110 mg/kg total). Another group of mice was treated with ISIS 116847 at 30 mg/kg once a day for one day. Each treatment group consisted of 3 animals. The mice were sacrificed 48 hrs following last administration. Liver tissues were homogenized and mRNA levels were quantitated using real-time PCR and normalized to RIBOGREEN as described herein. The results in Table 28 are presented as the average percent of PTEN mRNA levels for each treatment group, normalized to PBS-treated control and is denoted as “% UTC”. As illustrated, incorporation of C16 conjugate at position 8 showed comparable potency as position 1 at 110 mg/kg.

TABLE 28 Effect of C16 conjugated oligonucleotides on PTEN mRNA level Dosage UTC ISIS NO (mg/kg total) (%) Conjugate Saline 0 100 — 571032 110 22.05 C16, pos 8 580933 2.8 109.57 C16, pos 1 7 101.95 18 83.90 44 53.07 110 20.65 116847 30 44.06 5-10-5 MOE gapmer

Example 55 Modified Oligonucleotides Comprising C16 Conjugate Targeting PTEN In Vivo Study

An additional conjugated oligonucleotide was designed based on the parent oligomeric compound lacking the conjugate group, ISIS 522247. Since hydrolysis was observed at the amide bond between the alkyl linking group and the conjugate, the new conjugated oligonucleotide comprising a 5′-(E)-vinylphosphonate was designed by introducing a C16 conjugate group via a more stable carbamate linker at position 8 of the oligonucleotide. The newly designed conjugated oligonucleotide was tested and evaluated for their effects on PTEN mRNA levels in vivo. ISIS 522247 lacking a conjugate group and ISIS 571032 with a C16 conjugate group linked via an amide bond were included in the study for comparison.

The conjugated oligonucleotides were prepared using similar procedures as illustrated in Examples 12 and 17 and are described in Table 29. A subscript “s” between two nucleosides indicates a phosphorothioate internucleoside linkage (going 5′ to 3′). The absence of a subscript “s” between two nucleosides indicates a phosphodiester internucleoside linkage. A “Pv” at the 5′-end indicates a 5′-(E)-vinylphosphonate group, (PO(OH)₂(CH═CH—). Nucleosides followed by a subscript “f”, “m” or “e” are sugar modified nucleosides. A subscript “f” indicates a 2′-fluoro modified nucleoside, a subscript “m” indicates a 2′-O-methyl modified nucleoside and a subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside. Nucleosides with a subscript “C16” or “C16x” are shown below. Underlined nucleosides indicate the conjugate position. “NA” indicates not applicable.

Six week old BALB/C mice (Jackson Laboratory, Bar Harbor, Me.) were treated with ISIS 522247 at 25 mg/kg twice a day for two days (100 mg/kg total). Another group of mice was treated with ISIS 571032 or 589269 at 6, 14, 36 and 88 mg/kg once a day for one day. Each treatment group consisted of 4 animals. The mice were sacrificed 48 hrs following last administration. Liver tissues were homogenized and mRNA levels were quantitated using real-time PCR and normalized to RIBOGREEN as described herein. The results in Table 29 are presented as the average percent of PTEN mRNA levels for each treatment group normalized to PBS-treated control and is denoted as “% UTC”. As illustrated, treatment with the newly designed conjugated oligonucleotides showed an increase in potency in PTEN mRNA reduction as compared to treatment with the parent compound lacking a conjugate, ISIS 522247. In addition, incorporation of C16 conjugate at position 8 with a more stable carbamate linker exhibited comparable potency as compared to the amide linker.

The modified oligonucleotides were also evaluated for in vivo stability at dosage presented in Table 30. The tissue samples were collected and prepared using the same technique described in Example 25. Quantitative analysis of the oligonucleotides standard were performed by extracted ion chromatograms in the most abundant charge state (−4) using Chemstation software. The liver concentration (μg/g) of full length oligonucleotides comprising a 5′-terminal phosphonate group was measured by LC/MS and the results are presented below.

As illustrated in Table 30 and in FIG. 2, C16 conjugation increases liver accumulation of full-length oligonucleotides in a dose-dependent manner (ISIS 571032 and 589269). While the amide-linked C16 (ISIS 571032) hydrolyzed from the oligonucleotide, some fractions of carbamate-linked C16 conjugate, ISIS 589269 remained intact.

TABLE 28 Modified oligonucleotides comprising a C16 conjugate via an amide or a carbamate linker Conjugate/position ISIS NO Composition (5′ to 3′) counted from 5′ SEQ ID NO. 522247 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m)U_(fs)A_(m)A_(fs) No conjugate 6 U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) 571032 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m) U_(C16s) A_(m)A_(fs) C16 with amide 6 U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) linker, pos 8 589269 Pv-T_(es)U_(fs)A_(m)U_(fs)C_(m)U_(fs)A_(m) U_(C16xs) A_(m)A_(fs) C16 with carbamate 6 U_(m)G_(fs)A_(m)U_(fs)C_(ms)A_(fs)G_(ms)G_(fs)U_(ms)A_(es)A_(e) linker, pos 8

TABLE 29 Effect of modified oligonucleotides comprising a C16 conjugate via an amide or a carbamate linker on PTEN mRNA level Dosage UTC Conjugate/position ISIS NO (mg/kg total) (%) counted from 5′ SEQ ID NO. Saline 0 100 — — 522247 100 58.99 No conjugate 6 571032 6 102.27 C16 with amide 6 14 89.58 linker, pos 8 36 65.53 88 35.09 589269 6 101.69 C16 with carbamate 6 14 102.76 linker, pos 8 36 70.29 88 46.31

TABLE 30 Comparison of full-length modified oligonucleotides comprising a C16 conjugate via an amide or a carbamate linker in liver accumulation with ISIS 522247 in vivo Liver conc. of Liver conc. of Dosage full length full length ISIS (mg/kg/wk  ssRNA with loss ssRNA with NO total) of C16 (μg/g) C16 intact (μg/g) Conjugate Saline 0 N/A N/A — 522247 100 119.06 N/A No conjugate 571032 6 10.27 C16 conjugate C16 with an 14 23.82 completely amide linker 36 58.50 hydrolyzed 88 107.15 589269 6 6.22 2.8 C16 with a 14 15.93 16.79 carbamate 36 24.08 31.9 linker 88 39.76 83.98

Example 56 Hybridization-Dependent Nuclease ELISA Protocol

Liver samples (10-100 mg) were digested with 500 uL proteinase K digestion buffer (5U proteinase K (Sigma, St. Louis, Mo.)/1 mL Buffer G2 (Qiagen, Hilden, Germany)) for about 1 hour at 40° C. Standard curves were prepared with each analyte at 0.01 μM-5 μM in 500 μL control tissue homogenate (100 mg control liver/mL proteinase K digestion buffer) and digested 1 hr at 40° C. along with study samples. Study samples and standard curves were diluted 1:100 in blank liver digest and 25 uL hybridized with 475 uL 3 nM complementary hybridization probe that included a 5′ digoxigenin and 3′ biotin for 2 hrs at room temperature. 200 uL hybridization mix was added to NeutrAvidin-coated 96-well plates (Thermo, Rockford, Ill.) and incubated at room temperature for 1-2 hrs. NeutrAvidin plates were washed with 0.2% Tween 20 in Tris-buffered saline (TBST) and 300 uL 50-300 U/mL S1 nuclease (Life Technologies, Carlsbad, Calif.) was added and incubated at room temperature for 2 hrs. NeutrAvidin plates were washed with TBST and 200 uL 1:2000 anti-Digoxigenin-AP (Roche, Mannheim, Germany) was added and incubated for 1 hr. at room temperature. NeutrAvidin plates were washed with TBST and 200 uL Attophos (Promega, Madison Wis.) was added and fluorescence monitored (excitation 450/50, emission 580/50) using a SpectraMax Gemini microplate reader (Molecular Devices, Sunnyvale, Calif.). Catalysis of Attophos was stopped by addition of 100 uL saturated solution of disodium phosphate (25% Na₂HPO₄) before final quantitation of fluorescence on microplate reader.

Example 57 Modified Oligonucleotide Comprising C16 Conjugate Targeting PTEN In Vivo Study

ISIS 522247 and 543911 comprising a 5′-(E)-vinylphosphonate and C16 conjugate at position 8, as counted from the 5′-terminus was selected from the previous example and further evaluated for liver accumulation targeting PTEN in vivo.

Six week old BALB/C mice (Jackson Laboratory, Bar Harbor, Me.) were treated once a day for one day with ISIS 522247 or 543911 at 10, 50 or 100 mg/kg by intravenous (IV) injection. Another group of mice was treated with ISIS 522247 or 543911 twice a day for two days at 25 mg/kg (100 mg/kg total) by subcutaneous (SC) injection. Each treatment group consisted of 4 animals. The mice were sacrificed 48 hrs following last administration. Liver tissues were homogenized and analyzed using hybridization-dependent nuclease ELISA protocol as exemplified in Example 57 to determine the concentration of the conjugated oligonucleotide in the liver. The results are presented in Table 32 and FIG. 3. As illustrated, total concentration determined by ELISA showed more oligonucleotide in liver accumulation for C16 conjugate as compared to the parent oligonucleotide lacking the conjugate, ISIS 522247.

TABLE 32 Comparison of full-length modified oligonucleotides comprising a C16 conjugate in liver accumulation with ISIS 522247 in vivo Dosage Liver conc. of full (mg/kg/wk Mode of length ssRNA ISIS NO total) administration (μg/g) SEQ ID NO. 522247 10 IV  10.4 ± 10.1 6 50 47.09 ± 10.1 100 54.21 ± 14.6 100 SC 128.7 ± 21.0 543911 10 IV 35.8 ± 6.3 18 50  91.2 ± 28.4 100 241.82 ± 28.6  100 SC 297.47 ± 28.4  

We claim:
 1. A compound comprising an oligonucleotide consisting of 10-30 linked nucleosides and at least one conjugate group, wherein the 5′-terminal nucleoside of the oligonucleotide has Formula IV:

wherein: T₁ is

wherein: R_(a) and R_(c) are each, independently, OH, SH, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, amino or substituted amino; and R_(b) is O or S; T₂ is an internucleoside linking group linking the compound of Formula IV to the remainder of the oligonucleotide; Q₁ and Q₂ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy or N(R₃)(R₄); each R₃ and R₄ is, independently, H, C₁-C₆ alkyl or substituted C₁-C₆ alkyl; Bx is a heterocyclic base moiety; G is H, OH, halogen, a conjugate group, or O—[C(R₈)(R₉)]_(n)—[(C═O)_(m)—X₁]_(j)—Z; each R₈ and R₉ is, independently, H, halogen, C₁-C₆ alkyl or substituted C₁-C₆ alkyl; X₁ is O, S or N(E₁); Z is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or N(E₂)(E₃); E₁, E₂ and E₃ are each, independently, H, C₁-C₆ alkyl or substituted C₁-C₆ alkyl; n is from 1 to about 6; m is 0 or 1; j is 0 or 1; each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ₁, N(J₁)(J₂), ═NJ₁, SJ₁, N₃, CN, OC(═X₂)J₁, OC(═X₂)—N(J₁)(J₂) and C(═X₂)N(J₁)(J₂); X₂ is O, S or NJ₃; each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl; and provided that when j is 1 then Z is other than halogen or N(E₂)(E₃).
 2. The compound of claim 1, wherein the conjugate group is covalently attached to the oligonucleotide at a nucleoside at position 1, 2, 3, 4, 6, 7, 8, 9, 18, 19, 20, or 21 from the 5′-end of the oligonucleotide or at position 1, 2, 3, 12, 13, 4, 15, 17, 18, 19, 20, or 21 from the 3′-end of the oligonucleotide.
 3. The compound of claim 2, wherein the conjugate group is covalently attached to any of the 1 to 4 5′-most terminal nucleosides of the oligonucleotide.
 4. The compound of claim 2, wherein the conjugate group is covalently attached to the 5′-terminal nucleoside of the oligonucleotide.
 5. The compound of claim 2, wherein the conjugate group is covalently attached to the 8^(th) nucleoside from the 5′-terminal end of the oligonucleotide.
 6. The compound of claim 2, wherein the conjugate group is covalently attached to the 6^(th) nucleoside from the 5′-terminal end of the oligonucleotide.
 7. The compound of claim 2, wherein the conjugate group comprises C₂₀ alkyl.
 8. The compound of claim 2, wherein the conjugate group comprises C₁₆ alkyl.
 9. The compound of claim 2, wherein the conjugate group comprises C₁₀ alkyl.
 10. The compound of claim 2, wherein the conjugate group comprises a steroid.
 11. The compound of claim 2, wherein the conjugate group comprises cholesterol.
 12. The compound of claim 2, wherein the conjugate group comprises a carbohydrate.
 13. The compound of claim 2, wherein the conjugate group comprises N-acetylgalactosamine.
 14. The compound of claim 2, wherein each nucleoside is a modified nucleoside.
 15. The compound of claim 2, comprising at least one 2′-substituted nucleoside.
 16. The compound of claim 2, wherein the compound is single-stranded.
 17. The compound of claim 2, wherein each of the 6 to 9 3′-most terminal internucleoside linkages is a phosphorothioate linkage and each of the other internucleoside linkages is a phosphodiester linkage.
 18. The compound of claim 2, wherein each of the 6 to 9 3′-most terminal internucleoside linkages is a phosphorothioate linkage and each of the other internucleoside linkages is a phosphodiester or a phosphorothioate linkage.
 19. The compound of claim 2, wherein the conjugate group comprises C₁₀ to C₂₂ alkyl.
 20. The compound of claim 2, wherein the conjugate group comprises C₁₀ to C₂₂ alkenyl.
 21. The compound of claim 2, wherein the conjugate group comprises C₂₂ alkyl. 